Altered erythropoiesis and iron metabolism in carriers of thalassemia

The thalassemia syndromes (α‐ and β‐thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of α‐ or β‐globin chain production results in impaired red blood cell synthesis, anemia, and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis, and dysfunctional iron metabolism has not been investigated in both α‐thalassemia carriers (ATC) and β‐thalassemia carriers (BTC). Here, we demonstrate that ATC have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, BTC have several hematological parameters significantly different from controls, including increased soluble transferrin receptor and erythropoietin levels. These changes in both groups suggest an altered balance between erythropoiesis and iron metabolism. The index sTfR/log ferritin and (hepcidin/ferritin)/sTfR are, respectively, increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we showed in this study, for the first time in the literature, that thalassemia carriers have altered iron metabolism and erythropoiesis.     

Ineffective erythropoiesis and regulation of iron status in iron loading anaemias

The definition ‘iron loading anaemias’ encompasses a group of inherited and acquired anaemias characterized by ineffective erythropoiesis, low hepcidin levels, excessive iron absorption and secondary iron overload. Non‐transfusion‐dependent β‐thalassaemia is the paradigmatic example of these conditions that include dyserythropoietic and sideroblastic anaemias and some forms of myelodysplasia. Interrupting the vicious cycle between ineffective erythropoiesis and iron overload may be of therapeutic benefit in all these diseases. Induction of iron restriction by means of transferrin infusions, minihepcidins or manipulation of the hepcidin pathway prevents iron overload, redistributes iron from parenchymal cells to macrophage stores and partially controls anaemia in β‐thalassaemic mice. Inhibition of ineffective erythropoiesis by activin ligand traps improves anaemia and iron overload in the same models. Targeting iron loading or ineffective erythropoiesis shows promise in preclinical studies; activin ligand traps are in clinical trials with promising results and may be useful in patients with ineffective erythropoiesis.     

Relationship between transfusion regimen and suppression of erythropoiesis in β-thalassaemia major

In the management of β-thalassaemia major, different transfusion schemes are employed with baseline haemoglobin levels ranging from 8 to over 12 g/dl. We studied the relationship between transfusion regimen and suppression of erythropoiesis in 52 patients with β-thalassaemia major whose mean pretransfusion haemoglobin levels ranged from 8.6 to 10.9 g/dl. Multiple, regression analysis showed that serum transferrin receptor was the parameter more closely related to mean pretransfusion haemoglobin (r = -0.77, P < 0.001). As measured through serum transferrin receptor, erythroid activity was 1-2 times normal for pretransfusion haemoglobin levels between 10 and 11 g/dl, 1-4 times normal for levels from 9 to 10 g/dl, and 2-6 times normal for levels from 8.6 to 9 g/dl. Mean pretransfusion haemoglobin was also inversely related to serum erythropoietin (r = -0.72, P < 0.001), whereas it showed no or a weak relationship with Hb F, reticulocyte count, or circulating nucleated red cell count. This study suggests that serum transferrin receptor is a reliable indicator of suppression of erythropoiesis in β-thalassaemia major. On the basis of our findings, pretransfusion haemoglobin values of ≦ 9 g/dl should be adopted with caution, because these levels can be associated with an insufficient inhibition of erythroid marrow expansion. However, a transfusion programme, with a baseline haemoglobin of 9-10 g/dl, may provide enough suppression of erythropoiesis and allow a reduction in blood consumption as compared with the classic hyper- or supertransfusion schemes. Since fixed haemoglobin levels may not be the best target for transfusion treatment in all thalassaemic patients, assay of serum transferrin receptor may be helpful for individualizing the transfusion regimens.     

Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X-linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601 microgram/l, range 105-2855 microgram/l). Multiple regression analysis showed that 62% (P < 0.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.     

Advances in understanding erythropoiesis: evolving perspectives

Red blood cells (RBCs) are generated from haematopoietic stem and progenitor cells (HSPCs) through the step‐wise process of differentiation known as erythropoiesis. In this review, we discuss our current understanding of erythropoiesis and highlight recent advances in this field. During embryonic development, erythropoiesis occurs in three distinct waves comprising first, the yolk sac‐derived primitive RBCs, followed sequentially by the erythro‐myeloid progenitor (EMP) and HSPC‐derived definitive RBCs. Recent work has highlighted the complexity and variability that may exist in the hierarchical arrangement of progenitors responsible for erythropoiesis. Using recently defined cell surface markers, it is now possible to enrich for erythroid progenitors and precursors to a much greater extent than has been possible before. While a great deal of knowledge has been gained on erythropoiesis from model organisms, our understanding of this process is currently being refined through human genetic studies. Genes mutated in erythroid disorders can now be identified more rapidly by the use of next‐generation sequencing techniques. Genome‐wide association studies on erythroid traits in healthy populations have also revealed new modulators of erythropoiesis. All of these recent developments have significant promise not only for increasing our understanding of erythropoiesis, but also for improving our ability to intervene when RBC production is perturbed in disease.     

Serum hepcidin levels and muscle iron proteins in humans injected with low‐ or high‐dose erythropoietin

Inhibition of hepcidin expression by erythropoietic signals is of great physiological importance; however, the inhibitory pathways remain poorly understood. To investigate (i) the direct effect of erythropoietin (Epo) and (ii) the contribution of putative mediators on hepcidin repression, healthy volunteers were injected with a single dose of Epo, either low (63 IU/kg, n = 8) or high (400 IU/kg, n = 6). Low‐dose Epo provoked hepcidin down‐modulation within 24 h; the effect was not immediate as hepcidin circadian variations were still present following injection. High‐dose Epo induced no additional effect on the hepcidin response, that is hepcidin diurnal fluctuations were not abolished in spite of extremely high Epo levels. We did not find significant changes in putative mediators of hepcidin repression, such as transferrin saturation, soluble transferrin receptor, or growth differentiation factor 15. Furthermore, the potential hepcidin inhibitor, soluble hemojuvelin, was found unaltered by Epo stimulation. This finding was consistent with the absence of signs of iron deficiency observed at the level of skeletal muscle tissue. Our data suggest that hepcidin repression by erythropoietic signals in humans may not be controlled directly by Epo, but mediated by a still undefined factor.     

Serum erythropoietin and erythropoiesis in patients with myelodysplastic syndromes

Ineffective erythropoiesis is an early feature of the myelodysplastic syndromes (MDS), usually accompanying an hypercellular marrow. In a previous study, concentrations of serum erythropoietin (EPO) in MDS have been shown to correlate inversely both with haemoglobin concentration and with % bone marrow erythroblasts. We have measured erythroid production using a radioisotopic technique in 20 patients with MDS. Although haemoglobin concentration shows a weak inverse relationship with serum EPO concentration there is considerable variation in EPO concentration at a given haemoglobin level. There is no correlation between serum EPO and total erythroid production, though there is a weak correlation with effective erythropoiesis. The data suggests that control mechanisms of erythropoiesis in patients with MDS are complex.     

Indium chloride scanning: a comparison with iron as a tracer for erythropoiesis

The use of (¹¹¹In) indium for studying erythropoietic activity by scanning procedure has been investigated. ‘In scans have been compared with ⁵⁹Fe surface counts and ⁵²Fe scans in patients with various haematological conditions. Only in two of three patients with myelofibrosis was the activity of indium and iron similar; in all other patients, irrespective of the underlying disease, the two substances differed in their distribution in the body. By contrast, there was no discernible difference in the behaviour of ‘Fe and ⁵⁹Fe. It is concluded that indium cannot be relied on as an alternative to iron isotopes for ferrokinetic studies.     

The labile iron pool in human erythroid cells

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5·8-fold and 8·8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2·5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3·9-fold. We also show that in β-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.     

Assessment of erythropoiesis following renal transplantation

Abstract: Ten patients, who received cadaveric kidneys, were followed for 24 wk with serial measurements of serum erythropoietin (S-Epo), transferrin receptor (S-TfR) and iron variables. The mean pretransplant creatinine clearance was 8.2 (range 0–22) ml/min and the mean haemoglobin (Hb) level was 99±18.6 (range 66–124) g/l. Nine patients demonstrated a gradual increase in S-Epo levels, which reached a peak, and was accompanied by a parallel increase in S-TfR levels with a median lag period of 3 wk between both peaks. Hb correction followed the S-TfR peak after a second lag period (median 7 wk). Elevated S-Epo and S-TfR did not result in correction of anaemia in 1 patient due to impaired graft function. Within 4 months, S-Epo levels reached the normal range while TfR levels were higher than normal. Follow-up of iron status demonstrated the development of iron deficiency in 5 patients, which was corrected spontaneously. Improvement in erythropoiesis after renal transplantation seems to occur by means of expansion of the erythroid marrow, as detected by increasing S-TfR levels, subsequent to a S-Epo peak. This expansion precedes Hb normalization. A nonuraemic environment is probably a prerequisite for the correction of anaemia but not for the increase in S-Epo or S-TfR levels. Iron deficiency may occur after transplantation due to an increase in iron utilization.     

Iron metabolism in athletes – achieving a gold standard

Iron is an important mineral element required for diverse life processes. Its metabolism is almost synonymous to erythrocyte maintenance, erythropoiesis and erythrophagocytosis. Consequently, exercise exertion impacts significantly on red cell haematology. Here, the interactions between exercise and erythropoiesis are explored. Hepcidin, the peptide hormone that regulates systemic iron metabolism, decreases in response to erythropoiesis by facilitating increased iron efflux from ferroportin into circulation. However, during exercise, there is an alarming increase in the expression of hepcidin resulting in a negative iron balance in athletes. In this review, the confounding cause and effect scenarios of exercise, athlete training and haematology and hepcidin interactions are discussed.     

Iron Overload in a Non-Transfused Patient with Thalassaemia Intermedia

A patient with thalassaemia intermedia and haemosiderosis is reported. This patient did not receive transfusions or iron therapy. The iron absorption and the plasma iron turnover (PIT) were increased. Transfusions were carried out in order to decrease the amount of abnormal erythropoiesis. After that, the erythropoietin and PIT were decreased to normal levels and the iron absorption also returned to normal. The data presented suggest that increased erythropoiesis was responsible for the abnormaly high iron absorption and subsequently for the haemosiderosis of the patient presented.     

Microcytic anemia with iron malabsorption: An inherited disorder of iron metabolism

Two siblings were identified with severe hypoproliferative microcytic anemia and iron malabsorption, in the absence of any gastrointestinal disorder or blood loss. These children had severe microcytosis (MCV 48 fl, hemoglobin 7.5 g/dl) with decreased serum iron, elevated serum TIBC, and decreased serum ferritin, despite prolonged treatment with oral iron. An iron challenge study with an oral dose of 2 mg/kg elemental iron as ferrous sulfate documented iron malabsorption. After treatment with intravenous iron dextran, there was an absence of the expected reticulocytosis and only a partial correction of the hemoglobin, hematocrit, and microcytosis. The bone marrow was hypocellular with abnormal iron incorporation into erythroid precursor cells. This appears to be a rare form of inherited anemia characterized by iron malabsorption and disordered iron metabolism that only partially corrects after the administration of parenteral iron. These features resemble those found in the microcytic mouse (mk/mk), which also has severe microcytic anemia and iron malabsorption that partially responds to parenteral iron. © 1996 Wiley-Liss, Inc.     

Hepcidin--central regulator of iron metabolism

The knowledge about mammalian iron metabolism has advanced dramatically over the past decades. Studies of genetics, biochemistry and molecular biology allowed us the identification and characterization of many of the molecules involved in regulation of iron homeostasis. Important progresses were made after the discovery in 2000 of a small peptide--hepcidin--that has been proved to play a central role in orchestration on iron metabolism also providing a link between iron metabolism and inflammation and innate immunity. Hepcidin directly interacts with ferroportin (FPN), the only known mammalian iron exporter, which is expressed by enterocytes, macrophages and hepatocytes. The direct hepcidin-FPN interaction allows an adaptative response from the body in situations that alter normal iron homeostasis (hypoxia, anemia, iron deficiency, iron overload, and inflammation).     

Hepatic hypoxia-inducible factor-2 down-regulates hepcidin expression in mice through an erythropoietin-mediated increase in erythropoiesis

Background

Iron metabolism, regulated by the iron hormone hepcidin, and oxygen homeostasis, dependent on hypoxia-inducible factors, are strongly interconnected. We previously reported that in mice in which both liver hypoxia-inducible factors-1 and -2 are stabilized (the hepatocyte von Hippel-Lindau knockout mouse model), hepcidin expression was strongly repressed and we hypothesized that hypoxia-inducible factor-2 could be the major regulatory component contributing to the hepcidin down-regulation.

Design and Methods

We generated and analyzed hepatocyte-specific knockout mice harboring either hypoxia-inducible factor-2α deficiency (Hif2a knockout) or constitutive hypoxia-inducible factor-2α stabilization (Vhlh/Hif1a knockout) and ex vivo systems (primary hepatocyte cultures). Hif2a knockout mice were fed an iron-deficient diet for 2 months and Vhlh/Hif1a knockout mice were treated with neutralizing erythropoietin antibody.

Results

We demonstrated that hypoxia-inducible factor-2 is dispensable in hepcidin gene regulation in the context of an adaptive response to iron-deficiency anemia. However, its overexpression in the double Vhlh/Hif1a hepatocyte-specific knockout mice indirectly down-regulates hepcidin expression through increased erythropoiesis and erythropoietin production. Experiments in primary hepatocytes confirmed the non-autonomous role of hypoxia-inducible factor-2 in hepcidin regulation.

Conclusions

While our results indicate that hypoxia-inducible factor-2 is not directly involved in hepcidin repression, they highlight the contribution of hepatic hypoxia-inducible factor-2 to the repression of hepcidin through erythropoietin-mediated increased erythropoiesis, a result of potential clinical interest.     

Cl-IB-MECA inhibits human erythropoiesis

Candidate drugs are being sought for the suppression of human erythropoiesis. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide] is a derivative of adenosine that inhibits the growth of leukaemic cell lines. To determine the effects of Cl-IB-MECA upon erythropoiesis, studies were performed by using an ex vivo culture system of primary human CD34+ cells. Cl-IB-MECA suppressed erythroblast growth and maturation at doses >/=50 mumol/l through a mechanism of cell cycle inhibition and accumulation of cells in the G1/G0 phase. These findings demonstrate that Cl-IB-MECA inhibits human erythropoiesis, and suggest that further consideration of this drug is warranted for patients with erythrocytosis or polycythemia syndromes.     

Growth differentiation factor 15 in anaemia of chronic disease,iron deficiency anaemia and mixed type anaemia

Recently, the iron and erythropoiesis‐controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β‐thalassaemia patients, thereby increasing iron absorption despite iron overload. To access the diagnostic and pathogenic impact of GDF15 in inflammatory anaemia the association of GDF15 expression with serum iron parameters and hepcidin was studied in patients suffering from iron deficiency anaemia (IDA), anaemia of chronic disease (ACD) and ACD subjects with true iron deficiency (ACD/IDA). GDF15 was significantly increased in both ACD and ACD/IDA, but not in IDA subjects as compared to controls. In contrast, hepcidin levels were significantly lower in IDA and ACD/IDA subjects than in ACD patients . IDA and ACD/IDA, but not ACD, showed an association between GDF15 and soluble transferrin receptor, an indicator of iron requirement for erythropoiesis. However, GDF15 did not correlate to hepcidin in either patient group. While GDF15 levels were linked to the needs for erythropoiesis and iron homeostasis in IDA, the immunity‐driven increase of GDF15 may not primarily affect iron homeostasis and hepcidin expression. This indicates that other ACD‐related factors may overcome the regulatory effects of GDF15 on hepcidin expression during inflammation.     

Iron overload across the spectrum of non‐transfusion‐dependent thalassaemias: role of erythropoiesis,splenectomy and transfusions

Non‐transfusion‐dependent thalassaemias (NTDT ) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with β thalassaemia intermedia (n  = 95), haemoglobin (Hb) E/β thalassaemia (n  = 49) and Hb H syndromes (n  = 22). Liver iron concentration (LIC ), serum ferritin (SF ), transferrin saturation (TfSat) and non‐transferrin‐bound iron (NTBI ) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR ), labile plasma iron (LPI ) and nucleated red blood cells (NRBC s), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI , TfSat, NRBC s and SF relative to LIC , than non‐splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR , growth differentiation factor‐15, NRBC s) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI , LPI and NRBC s. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra‐hepatic iron distribution are more likely in previously transfused, splenectomised and iron‐overloaded NTDT patients with TfSat >70%.