Yelena Ginzburg

Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice

In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV), or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin messenger RNA (mRNA) responsiveness to short and long‐term iron challenge in iron‐depleted Hfe, Tfr2, Hjv, and Bmp6 mutant mice. After 1‐day (acute) iron challenge, Hfe−/− mice showed a smaller hepcidin increase than their wild‐type strain‐matched controls, Bmp6−/− mice showed nearly no increase, and Tfr2 and Hjv mutant mice showed no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21‐day (chronic) iron challenge, Hfe and Tfr2 mutant mice increased hepcidin expression to nearly wild‐type levels, but a blunted increase of hepcidin was seen in Bmp6−/− and Hjv−/− mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (except Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV, BMP6, and, to a lesser extent, HFE are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2, and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading. (HEPATOLOGY 2011;)

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Transferrin therapy ameliorates disease in [beta]-thalassemic mice

Individuals with β-thalassemia develop progressive systemic iron overload, resulting in high morbidity and mortality. These complications are caused by labile plasma iron, which is taken up by parenchymal cells in a dysregulated manner; in contrast, erythropoiesis depends on transferrin-bound iron uptake via the transferrin receptor. We hypothesized that the ineffective erythropoiesis and anemia observed in β-thalassemia might be ameliorated by increasing the amount of circulating transferrin. We tested the ability of transferrin injections to modulate iron metabolism and erythropoiesis in Hbbth1/th1 mice, an experimental model of β-thalassemia. Injected transferrin reversed or markedly improved the thalassemia phenotype in these mice. Specifically, transferrin injections normalized labile plasma iron concentrations, increased hepcidin expression, normalized red blood cell survival and increased hemoglobin production; this treatment concomitantly decreased reticulocytosis, erythropoietin abundance and splenomegaly. These results indicate that transferrin is a limiting factor contributing to anemia in these mice and suggest that transferrin therapy might be beneficial in human β-thalassemia.

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas–Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis

Terminal erythroid differentiation is the process during which proerythroblasts differentiate to produce enucleated reticulocytes. Although it is well established that during murine erythropoiesis in vivo, 1 proerythroblast undergoes 3 mitosis to generate sequentially 2 basophilic, 4 polychromatic, and 8 orthochromatic erythroblasts, currently there is no method to quantitatively monitor this highly regulated process. Here we outline a method that distinguishes each distinct stage of erythroid differentiation in cells from mouse bone marrow and spleen based on expression levels of TER119, CD44, and cell size. Quantitative analysis revealed that the ratio of proerythroblasts:basophilic:polychromatic:orthromatic erythroblasts follows the expected 1:2:4:8 ratio, reflecting the physiologic progression of terminal erythroid differentiation in normal mice. Moreover, in 2 stress erythropoiesis mouse models, phlebotomy-induced acute anemia and chronic hemolytic anemia because of 4.1R deficiency, the ratio of these erythroblast populations remains the same as that of wild-type bone marrow. In contrast, in anemic β-thalassemia intermedia mice, there is altered progression which is restored to normal by transferrin treatment which was previously shown to ameliorate the anemic phenotype. The means to quantitate in vivo murine erythropoiesis using our approach will probably have broad application in the study of altered erythropoiesis in various red cell disorders.

Prolonged red cell storage before transfusion increases extravascular hemolysis

BACKGROUND. Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS. Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS. Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS. After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal. REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING. NIH grant HL115557 and UL1 TR000040.

Impact of iron overload and potential benefit from iron chelation in low-risk myelodysplastic syndrome

Myelodysplastic syndromes (MDSs) are a group of heterogeneous clonal bone marrow disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and potential for malignant transformation. Lower/intermediate-risk MDSs are associated with longer survival and high red blood cell (RBC) transfusion requirements resulting in secondary iron overload. Recent data suggest that markers of iron overload portend a relatively poor prognosis, and retrospective analysis demonstrates that iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. New data provide concrete evidence of irons adverse effects on erythroid precursors in vitro and in vivo. Renewed interest in the iron field was heralded by the discovery of hepcidin, the main serum peptide hormone negative regulator of body iron. Evidence from β-thalassemia suggests that regulation of hepcidin by erythropoiesis dominates regulation by iron. Because iron overload develops in some MDS patients who do not require RBC transfusions, the suppressive effect of ineffective erythropoiesis on hepcidin may also play a role in iron overload. We anticipate that additional novel tools for measuring iron overload and a molecular-mechanism-driven description of MDS subtypes will provide a deeper understanding of how iron metabolism and erythropoiesis intersect in MDSs and improve clinical management of this patient population.

Iron overload in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of disorders characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Iron overload results from high transfusion requirements and retrospective studies have shown it to be associated with relatively poor survival in a subset of the low risk patients. Recent discoveries have led to the identification of hepcidin as a key regulator of iron metabolism and to the association of non-transferrin bound iron moieties, such as labile plasma iron, with the end organ damage in iron overload states. Currently, there is limited data in evaluating the role of iron chelators in MDS and data from studies in Thalassemia and hemachromostosis have been used to predict ferritin levels above 1000 – 2500 ng/mL and history of 20 blood transfusions as clinical end points for considering iron chelation in MDS. Deferoxamine and deferasirox, the two iron chelators approved for use in the US, have shown efficacy in reducing iron overload in MDS in retrospective studies are now being evaluated for effects on overall survival in prospective studies. On the basis of retrospective data, it is reasonable to offer iron chelation to the lower risk MDS patients requiring frequent transfusions, while monitoring for specific adverse affects in patients on treatment.

Crosstalk between Iron Metabolism and Erythropoiesis.

Iron metabolism and erythropoiesis are inextricably linked. The majority of iron extracted from circulation daily is used for hemoglobin synthesis. In the last 15 years, major advances have been made in understanding the pathways regulating iron metabolism. Hepcidin is a key regulator of iron absorption and recycling and is itself regulated by erythropoiesis. While several viable candidates have been proposed, elucidating the “erythroid regulator” of hepcidin continues to generate significant experimental activity in the field. Although the mechanism responsible for sensing iron demand for erythropoiesis is still incompletely understood, evaluating diseases in which disordered erythropoiesis and/or iron metabolism are showcased has resulted in a more robust appreciation of potential candidates coordinated erythroid iron demand with regulators of iron supply. We present data drawn from four different conditions—iron deficiency, congenital hypotransferrinemia, beta-thalassemia, and hereditary hemochromatosis—both in human and non-human models of disease, together suggesting that erythroid iron demand exerts a stronger influence on circulating iron supply than systemic iron stores. Greater understanding of the interplay between the key factors involved in the regulation of iron metabolism and erythropoiesis will help develop more effective therapies for disorders of iron overload, iron deficiency, and hemoglobin synthesis.

Minihepcidin peptides as disease modifiers in mice affected by β-thalassemia and polycythemia vera

In β-thalassemia and polycythemia vera (PV), disordered erythropoiesis triggers severe pathophysiological manifestations. β-Thalassemia is characterized by ineffective erythropoiesis, reduced production of erythrocytes, anemia, and iron overload and PV by erythrocytosis and thrombosis. Minihepcidins are hepcidin agonists that have been previously shown to prevent iron overload in murine models of hemochromatosis and induce iron-restricted erythropoiesis at higher doses. Here, we show that in young Hbb(th3/+) mice, which serve as a model of untransfused β-thalassemia, minihepcidin ameliorates ineffective erythropoiesis, anemia, and iron overload. In older mice with untransfused β-thalassemia, minihepcidin improves erythropoiesis and does not alter the beneficial effect of the iron chelator deferiprone on iron overload. In PV mice that express the orthologous JAK2 mutation causing human PV, administration of minihepcidin significantly reduces splenomegaly and normalizes hematocrit levels. These studies indicate that drug-like minihepcidins have a potential as future therapeutics for untransfused β-thalassemia and PV.