Iron Overload In Β-thalassemia Research Articles

A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in β-thalassemic mice

AbstractIron plays a major role in the deterioration of β-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in β-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in β-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the β-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of β-thalassemia.

Baicalein Protects Against Iron Overload Induced Liver Dysfunction in Thalassemic Mice

Liver iron overload is a common and serious organ injury in β-thalassemia patients. Ferroptosis has been shown to play a crucial role in the pathological injury in iron overloaded hepatocytes. In our study, we focused on the protective effects of baicalein, a natural, active flavone extracted from an herb used in traditional Chinese medicine against RSL3-induced ferroptosis in hepatocytes and high iron diet (HID) induced liver iron-overload in a murine β654-thalassemia model. In vitro, the effects of baicalein on RSL3-induced hepatocyte ferroptosis were examined by testing ferroptosis related genes, protein, and the GSH, MDA, iron ion level by RT-PCR, Western blot and the commercial kits respectively. The anti-iron overload injury effects of baicalein were assessed in a β654-thalassemia mouse model of high iron diet induced liver injury by the same method In vitro experiments. Here, we found that baicalein could reverse cell ferroptosis in hepatocytes treated with RSL3. Importantly, changes in the iron ion content, and MDA and GSH levels in β654-thalassemia mouse livers were significantly restored by baicalein. Mechanistically, baicalein may activated the NRF2 antioxidant pathway and increased the expression of GPX4 in vivo and In vitro. We concluded that baicalein is a potential therapeutic drug for the treatment of iron overload in β-thalassemia.

Erythroferrone Overexpression Impairs Survival and Greatly Increases Tissue Iron Loading in a Mouse Model of β-Thalassemia

Beta-thalassemia, caused by mutations in the β-globin gene, is characterized by ineffective erythropoiesis, anemia and associated multi-organ dysfunction. Like in other anemias with ineffective erythropoiesis, the erythroid hormone erythroferrone (ERFE) is pathologically overproduced. ERFE functions by suppressing the master iron regulatory hormone hepcidin, and the chronic suppression of hepcidin in β-thalassemia results in excessive iron absorption and iron overload, even in patients who do not receive erythrocyte transfusions. A major obstacle in studying the role of ERFE in β-thalassemia has been the lack of a mouse model that represents the wide range of ERFE concentrations observed in human patients (Ganz et al, Blood 2017). Our lab generated ERFE-transgenic mice with a range of ERFE overexpression (Coffey et al, Blood 2022) and showed that they developed dose-dependent iron overload and relative hepcidin deficiency. In the current study, the mid-range ERFE-overexpressing E(M) mice were crossed with HbbTh3/+ (Th3) mice, a widely used model of β-thalassemia, to generate mice with a single copy of both transgenes (T-E(M)), as well as littermate controls (Th3, WT, E(M)), to analyze the effect of ERFE on the pathophysiology of ineffective erythropoiesis. The offspring had a noticeably skewed genotypic ratio, with T-E(M) pups making up only <9% of surviving pups compared to 25% expected for each genotype. At embryonic day 18.5, however, there was an equal distribution of all expected genotypes, suggesting that the mortality in T-E(M) pups is postnatal. Surviving T-E(M) mice were analyzed at 16 weeks of age. They had similar erythrocyte parameters to the Th3 littermates , with the expected decrease in red blood cell count, hemoglobin and mean corpuscular volume. Despite the similarities in blood counts, we discovered a synergistic effect of the ERFE and Th3 transgenes on ERFE production in the T-E(M) mice as indicated by increased serum ERFE levels (Figure 1a) and increased Erfe mRNA levels in the bone marrow and spleen compared with both E(M) and Th3 littermates. The dramatic increase in serum ERFE in T-E(M) mice may result from multiplicative effects of erythroblast expansion in Th3 mice and transgenic ERFE overexpression in individual erythroblasts of T-E(M) mice. The large range of ERFE concentrations in human patients may also reflect the combined effects of erythroblast number and ERFE overproduction by erythropoietin-stimulated thalassemic erythroblasts, both of which vary depending on patient genetics. T-E(M) mice also have significantly larger spleens compared to Th3 littermates, suggesting that ERFE overexpression contributes to increased extramedullary hematopoiesis in T-E(M) spleens. In addition, T-E(M) mice had significantly higher iron levels in the liver (Figure 1b) as well as serum and kidney compared to Th3 littermates. Coupled with the inappropriately low serum hepcidin levels (compared to liver iron levels) in T-E(M) mice, even when compared to E(M) and Th3 littermates, these data indicate that excessive ERFE further increases iron loading in a mouse model of β-thalassemia. Our data demonstrate that elevated ERFE levels contribute to the pathogenesis of β-thalassemia by decreasing postnatal survival, increasing iron overload, and increasing extramedullary hematopoiesis. These results suggest that neutralizing excessive ERFE levels may have therapeutic benefits for postnatal development and prevention of iron overload in β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of iron homeostasis by hepatocyte TfR1 requires HFE and contributes to hepcidin suppression in β-thalassemia

AbstractTransferrin receptor 1 (TfR1) performs a critical role in cellular iron uptake. Hepatocyte TfR1 is also proposed to influence systemic iron homeostasis by interacting with the hemochromatosis protein HFE to regulate hepcidin production. Here, we generated hepatocyte Tfrc knockout mice (Tfrcfl/fl;Alb-Cre+), either alone or together with Hfe knockout or β-thalassemia, to investigate the extent to which hepatocyte TfR1 function depends on HFE, whether hepatocyte TfR1 impacts hepcidin regulation by serum iron and erythropoietic signals, and its contribution to hepcidin suppression and iron overload in β-thalassemia. Compared with Tfrcfl/fl;Alb-Cre− controls, Tfrcfl/fl;Alb-Cre+ mice displayed reduced serum and liver iron; mildly reduced hematocrit, mean cell hemoglobin, and mean cell volume; increased erythropoietin and erythroferrone; and unchanged hepcidin levels that were inappropriately high relative to serum iron, liver iron, and erythroferrone levels. However, ablation of hepatocyte Tfrc had no impact on iron phenotype in Hfe knockout mice. Tfrcfl/fl;Alb-Cre+ mice also displayed a greater induction of hepcidin by serum iron compared with Tfrcfl/fl;Alb-Cre− controls. Finally, although acute erythropoietin injection similarly reduced hepcidin in Tfrcfl/fl;Alb-Cre+ and Tfrcfl/fl;Alb-Cre− mice, ablation of hepatocyte Tfrc in a mouse model of β-thalassemia intermedia ameliorated hepcidin deficiency and liver iron loading. Together, our data suggest that the major nonredundant function of hepatocyte TfR1 in iron homeostasis is to interact with HFE to regulate hepcidin. This regulatory pathway is modulated by serum iron and contributes to hepcidin suppression and iron overload in murine β-thalassemia.

Role of malonaldehyde as a surrogate biomarker for iron overload in the β-thalassemia patient: A systematic meta-analysis

Malonaldehyde (MDA), a biomarker of oxidative stress is found to be increased in many individuals suffering from β-thalassemia. This may possibly be attributed to iron overload that is seen occurring in varying degrees in the condition as evident from increased ferritin (the storage form of body iron) levels in afflicted individuals. This study hypothesized that MDA may be used as a surrogate marker for iron overload in β-thalassemia. We performed a systematic review and meta-analysis of case-control studies for both parameters. The mean difference (MD) and confidence interval at 95% was calculated using a random effect model after exclusion of unsuitable data and study imputed of bias by trim-fill analysis. The study subjects were divided into two groups; transfusion-dependent β-thalassemia (TDT) and non-transfusion-dependent β-thalassemia (NTDT). The study population for MDA comprised of 2001 candidates (1159 Cases and 842 Controls) and 429 individuals (289 Cases and 140 Controls) respectively, for TDT and NTDT groups. While 1332 (803 Cases vs. 529 Controls) and 342 (234 Cases vs. 108 Controls) candidates were included in the analysis of ferritin in the aforesaid two groups. It was found that MDA levels were significantly increased in TDT [MD 2.221 (1.80-2.646), p<0.001] as well as in NTDT [MD 1.89 (1.20-2.581), p<0.001] when compared with normal individuals. Ferritin level is also found to be significantly increased in these two groups: in TDT [MD 2.288 (1.883-2.693), p<0.001] and in NTDT [MD 1.946 (0.883-3.009), p<0.001] when compared with normal individuals. In conclusion, it is seen that all β-thalassemia patients irrespective of blood transfusion dependency suffer from the curse of iron overload-induced oxidative stress.

The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia.

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia.

Additive Effects of Decreased TfR1 and Ablated Erfe Improve Both Ineffective Erythropoiesis and Iron Overload in β-Thalassemic Mice

Patients with β-thalassemias manifest anemia, ineffective erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as ineffective erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness. DisclosuresGanz:Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.

The role of erythroferrone in iron metabolism: From experimental results to pathogenesis

In case of erythropoiesis, body iron needs to increase to enable the production of new red blood cells. In the 1950s, the observation of an increased digestive iron absorption in the case of phlebotomies had led to propose the existence of an "erythroid factor", which regulate the availability of iron for erythropoiesis in this situation. The factor regulating iron stores has been identified in 2000 to be hepcidin. Recently, in 2014, a new factor was discovered, which regulates iron metabolism, independently of iron stores and responds to the increased requirements for iron after stimulation of erythropoiesis by erythropoietin. This factor has been referred to as erythroferrone. Thus, the regulation of iron stores depends on hepcidin, while the adaptation mechanisms of iron availability in case of anemia, are mediated by an erythroid factor that could be erythroferrone. This review summarizes the current knowledge on the role of erythroferrone in iron metabolism, starting from experimental results, obtained mainly on mouse models, and related to iron overload in β-thalassemia, iron disturbances during anemia of chronic diseases and chronic renal failure. These results will have to be compared with those obtained in humans, as soon as a reliable assay for human erythroferrone is available. From a clinical point of view, erythroferrone could become a useful biological marker of iron metabolism and a therapeutic target.

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Role of Erythroferrone in the Pathophysiology of Common Anemias

Introduction: We recently described the erythroid hormone erythroferrone (ERFE) as a critical regulator of hepcidin production during increased erythropoietic activity. Both anemia of inflammation (AI) and beta-thalassemia are associated with elevated levels of erythropoietin, the signal directing ERFE expression, suggesting that ERFE may play a role in the pathogenesis of these disorders. Indeed, Erfe mRNA expression was highly increased in the bone marrow and the spleen of a mouse model of β-thalassemia Hbbth3/+ (Th3/+). We therefore investigated the role of erythroferrone in hepcidin regulation in anemia of inflammation and β-thalassemia. We report that ERFE contributes to the recovery from anemia of inflammation and may be a hepcidin-suppressive factor responsible for iron accumulation in thalassemia.Methods: We explored the role of ERFE in anemia of inflammation using the heat-killed Brucellaabortus (BA) mouse model of AI. Hepcidin regulation and recovery from anemia was compared between wild-type and Erfe-deficient mice. To determine whether ERFE is the hepcidin-suppressive factor in thalassemia, we generated Erfe-/-/Th3/+ double mutant mice and compared them to their littermate WT, Erfe-/- and Th3/+ mice. Mice were compared at 3, 6 and 12 weeks of age.Results: Compared to wild-type mice, Erfe-deficient mice did not appropriately decrease hepcidin production during recovery from AI and exhibited greater severity of anemia (figure). These data suggest that compensatory hepcidin suppression during the recovery phase of AI is mediated by ERFE. However, in mice this defect was partly compensated by prolonged stimulation of erythropoiesis and higher reticulocytosis resulting in Erfe-deficient mice reaching wild-type hemoglobin levels by day 28 (figure). [Display omitted] At the other end of the spectrum, we showed that thalassemic mice exhibited greatly increased expression of Erfe mRNA in the bone marrow and the spleen. Ablation of Erfe in Th3/+ mice restored normal hepcidin levels and significantly reduced serum iron concentration and hepatic iron overload at 6 weeks of age. Comparison of Erfe-/- / Th3/+ and Th3/+ mice at 3, 6 and 12 weeks of age did not show any difference in the severity of the anemia in absence of ERFE suggesting that ERFE regulates hepcidin expression but its absence did not cause iron restriction and did not ameliorate ineffective erythropoiesis.Conclusion: ERFE is a critical regulator of hepcidin expression during recovery from AI and ERFE agonists may alleviate iron restriction in AI. ERFE may also be the factor responsible for hepcidin suppression and secondary iron overload in β-thalassemia. DisclosuresGanz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.

Exogenous Apo-Transferrin Increases Monoferric Transferrin, Decreasing Cytosolic Iron Uptake and Heme and Globin Synthesis in β-Thalassemic Mice

β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P<0.0001), and an equal concentration apoTf:holoTf mixture results in intermediate binding between apoTf and holoTf (P=0.004). We also demonstrate a dose dependent decrease in cytosolic iron (P<0.001) and heme (P<0.01) in MEL cells treated with escalating concentrations of apoTf, an effect abrogated by the additional of DFO, an iron chelator; urea gel of MEL cell supernatants reveals that monoferric Tf increases with increasing doses of apoTf and decreased in concurrently DFO treated cells (Figure 1). These findings together suggest that changes in iron uptake result from increased monoferric Tf (rather than competition of apoTf for TfR1 binding sites). In addition, in vivo experiments demonstrate a decrease in heme concentration (56 vs. 67 uM, P<0.0001) in circulating RBCs as well as α-globin (1.3 vs. 3.3-fold, P=0.009) and β-globin (Figure 2) mRNA expression in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice. As heme is known to regulate globin expression through transcriptional and translational routes, we evaluate bach1, heme oxygenase 1 (HO-1), and heme-regulated eIF2α kinase (HRI). We demonstrate no difference in bach1 and HO-1 mRNA expression but a significant and unexpected decrease in HRI expression (1.0 vs. 2.8-fold, P=0.01) in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice (similar findings in all stages of terminal erythroid differentiation), suggesting that HRI is regulated by mechanisms independent of cellular iron and heme. Furthermore, western blots of HRI and its target eIF2α (total and phosphorylated) are also normalized in sorted bone marrow erythroid precursors from apoTf- vs. PBS-treated th1/th1 mice (Figure 3). Taken together, our data suggests that exogenous apoTf in th1/th1 mice results in decreased cytosolic iron and heme as well as α- and β-globin synthesis as a consequence of increased monoferric Tf in circulation and provides a novel system to explore the regulation of globin in β-thalassemia and how iron deficiency benefits this disease. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy of deferasirox in reducing and preventing cardiac iron overload in β-thalassemia

Cardiac iron overload causes most deaths in beta-thalassemia major. The efficacy of deferasirox in reducing or preventing cardiac iron overload was assessed in 192 patients with beta-thalassemia in a 1-year prospective, multicenter study. The cardiac iron reduction arm (n = 114) included patients with magnetic resonance myocardial T2* from 5 to 20 ms (indicating cardiac siderosis), left ventricular ejection fraction (LVEF) of 56% or more, serum ferritin more than 2500 ng/mL, liver iron concentration more than 10 mg Fe/g dry weight, and more than 50 transfused blood units. The prevention arm (n = 78) included otherwise eligible patients whose myocardial T2* was 20 ms or more. The primary end point was the change in myocardial T2* at 1 year. In the cardiac iron reduction arm, the mean deferasirox dose was 32.6 mg/kg per day. Myocardial T2* (geometric mean +/- coefficient of variation) improved from a baseline of 11.2 ms (+/- 40.5%) to 12.9 ms (+/- 49.5%) (+16%; P < .001). LVEF (mean +/- SD) was unchanged: 67.4 (+/- 5.7%) to 67.0 (+/- 6.0%) (-0.3%; P = .53). In the prevention arm, baseline myocardial T2* was unchanged from baseline of 32.0 ms (+/- 25.6%) to 32.5 ms (+/- 25.1%) (+2%; P = .57) and LVEF increased from baseline 67.7 (+/- 4.7%) to 69.6 (+/- 4.5%) (+1.8%; P < .001). This prospective study shows that deferasirox is effective in removing and preventing myocardial iron accumulation. This study is registered at http://clinicaltrials.gov as NCT00171821.

Crosstalk between Erythropoiesis and Iron Metabolism

Oxygen delivery is essential to sustain all vital functions. Vertebrate organisms have, therefore, evolved sophisticated and tightly regulated mechanisms to ensure continuous red cell synthesis. Chronic or acute blood losses trigger multiple feedback mechanisms aimed at increasing erythropoiesis and minimizing the damage associated with profound anemia and hypoxia. Iron is an element of paramount importance for erythropoiesis, being an essential part of the hemoglobin metalloprotein that ensures oxygen absorption, transport, and delivery. Therefore, it is not surprising that iron absorption and transport are closely linked to the demands of erythropoiesis. However, the mechanisms underlying this interconnected relationship have only recently started being understood. The focus of this special issue is on iron metabolism, erythropoiesis, and their close association, with a special emphasis on proteins regulating iron metabolism and how erythropoiesis affects their expression. The mechanistic view will be integrated with the clinical observations in disease such as myelodysplastic syndromes, sickle cell anemia, Diamond-Blackfan anemia, and anemia of inflammation. And finally, in light of these new discoveries, new potential therapies for well-known disorders such as beta-thalassemia will be discussed. In the last decade, new discoveries have fueled the field of iron metabolism. The papers by H. Li and Y. Z. Ginzburg[1],E. Nemeth [2], L. Cianetti et al. [3], and T. Tanno and J. L. Miller [4] provide a comprehensive and updated overview of the proteins involved in regulating dietary iron absorption, plasma iron concentrations, tissue iron distribution, and erythropoiesis. The review by H. Li and Y. Z. Ginzburg [1] introduces the mechanisms of crosstalk between iron and erythropoiesis and their function in different diseases. E. Nemeth [2] describes the role of the peptide hormone hepcidin in iron physiology and in many iron-related disorders and highlights the potential of hepcidin agonists and antagonist as future therapeutic tools for iron disorders. The paper by L. Cianetti et al. [3] describes the hepcidin receptor and iron transporter ferroportin and its role during normal and pathological erythroid differentiation. The paper by T. Tanno and J. L. Miller [4] describes the relationship between iron regulation and ineffective erythropoiesis, focusing on potential mechanisms for pathological iron overloading. Improved knowledge on proteins that interconnect iron metabolism and erythropoiesis is opening new prospective to develop new therapies, such as in beta-thalassemia. The review by L. Melchiori et al. [5] provides important evidence that use of Jak2 inhibitors or hepcidin agonists may ameliorate the abnormal erythropoiesis and iron overload in β-thalassemia. The interdependence of erythropoiesis and iron regulation extends beyond normal metabolism to include several pathological conditions. Diseases that are primarily associated with erythroid defects often lead to iron overload, maldistribution, or deficiency. The reviews by E. Messa et al. [6] and by M. A. Badawi et al. [7] summarize the historical progress and recent clinical developments using iron chelation in patients with myelodysplastic syndromes. Similarly, the paper by R. Raghupathy et al. [8] provides an overview of transfusional iron overload patients with sickle cell disease, highlighting how to prevent iron overload in this disorder. Diamond-Blackfan anemia is a rare congenital pure red-cell aplasia that presents during infancy. D. Chiabrando and E. Tolosano's [9] review focused upon heme and ribosomal biogenesis in this disease and why deficiency of the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1) may cause a related phenotype. Finally, the review by E. A. Price and S. L. Schrier [10] provides a comprehensive and updated overview of anemia of inflammation, focusing on many of key clinical issues that remain to be clarified, such as understanding of mechanisms of anemia of inflammation in “noninflammatory” diseases, optimal methods for distinguishing anemia of inflammation from iron deficiency anemia, and understanding the contributory role of various pathologic mechanisms in individual human diseases that lead to anemia of inflammation. We hope that this special issue will stimulate curiosity, interest, and new research directions in exploring the field of iron and erythropoiesis, with the ultimate goal to develop new drugs and improve the management of disorders associated with altered iron metabolism, abnormal erythropoiesis, and anemia of inflammation. Stefano Rivella Elizabeta Nemeth Jeffery Lynn Miller

The Uses and Differences of R2 and R2* in the Determination of Iron Overload in Iron Loaded Thalassemia Patients

Early attempts to use magnetic resonance imaging (MRI) for assessing iron overload in β-thalassemia (thal) patients began more than 20 years ago. With advances in MRI, more quantitative efforts focused on measuring transverse relaxation time rates (R2 and R2*) of the liver and/or myocardium. Recently, calibration curves of R2 and R2* were reported that allowed one to determine the absolute concentrations of iron in the liver, provided that R2 and R2* were determined with the same technique. The difficulty of obtaining sufficient myocardium biopsy samples has prevented similar calibration curves being reported for the myocardium. Preliminary data indicate that liver and myocardium R2* vs. R2 plots are similar over a large range of R2* and R2 values. Obviously, myocardium biopsy samples are needed to confirm whether myocardium R2* and R2 plots vs. iron concentration are similar to those published for the liver. The various methods for determining R2 and R2* are discussed. It is suggested to use both R2* and R2 for assessing iron overload in the liver and myocardium.

Ineffective Erythropoiesis in β-Thalassemia Is Characterized by Increased Iron Absorption Mediated by down Regulation of Hepcidin and up Regulation of Ferroportin.

Progressive iron overload occurs in β-thalassemia as a result of increased gastrointestinal absorption. Our goal is to investigate the relationship between ineffective erythropoiesis (IE), iron-related genes and organ iron distribution in mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. The th3/th3 mice die in 8 weeks due to severe anemia but can be rescued by transfusion therapy. We analyzed up to 90 animals at 2, 5 and 12 months, as appropriate. We monitored various hematological parameters, tissue iron content and quantitative-PCR levels of Hamp, Fpn1, Smad4, Cebpa, Hfe, Tfr1 and other genes involved in iron metabolism in liver, spleen, kidney, heart and duodenum. At 2 months, th3/th3 mice had the highest total body iron content and highest degree of IE. The total iron was 53.6±21.0, 406.1±156.1, 657.7±40.3 μg in the spleen, and 107.5±35.7, 208.5±24.9 and 1298.7±427.5 μg in the liver of +/+, th3/+ and th3/th3, respectively (n≥5 per genotype). However, if the organ size was not taken in account, the iron concentration in the spleen of th3/+ was higher, in average, than that of th3/th3 mice (3.8±1.5 and 2.9±0.5 μg/mg), while in the liver was the opposite (0.6±0.1 and 5.1±2.0 μg/mg of dry weight, P<0.001). Heme and non-heme iron analyses provided similar results. Surprisingly, the distribution of iron within organs also differed. In th3/+ mice, the hepatic iron was almost exclusively located in Kupffer cells, whereas in th3/th3 mice in parenchymal cells. Our data suggest that Hamp is responsible for the increased iron absorption, being reduced to 20% and 70% in 2 month-old th3/+ and th3/th3 mice compared to +/+ animals (P<0.001). Hfe was reduced by 50% (P<0.05) in the liver of the animals that expressed low Hamp levels, indicating that Hfe could be directly responsible for Hamp regulation or share the same regulatory pathway. Low levels of Smad4 and Cebpa were observed only in the liver of mice with the lowest Hamp expression (P<0.05), indicating that these proteins might contribute to further decreased Hamp synthesis. In addition, while Tfr1 in th3/+ mice was 40% lower in the liver, it was up-regulated (400%) in th3/th3 mice (P<0.001), which may explain why iron is increased more in the liver of th3/th3 mice. In 5 and 12 month-old th3/+ mice, the surprising observation was the normal expression level of Hamp. However, in the duodenum, the Fpn1 RNA and protein levels were augmented (300%, P<0.001). In transfused th3/+ and th3/th3 animals, Hamp, Hfe, Cbpa and Smad4 expression levels were normalized or increased, while Tfr1 was down-regulated in both groups, which may explain the increased splenic iron deposition in these animals. Our data suggest that IE, together with the relative expression levels of Hamp and Tfr1, is largely responsible for the organ iron overload observed in young thalassemic mice. However, in older mice, it is the increase of Fpn1 levels in the duodenum that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the genesis of iron overload in β-thalassemia.

Oral iron chelators for clinical use

The clinical problems connected to iron overload in β-thalassemia are briefly summarized and the need for finding proper iron chelators is presented. The biological, pharmacological and clinical requirements for an ideal iron chelator are discussed. Some chemical considerations on iron chelators as well as a survey on chemical compounds tested up to now are presented.

Plasma Chitotriosidase Activity in Patients with β-Thalassemia

ABSTRACT: Chitotriosidase, a macrophage marker, which is extremely increased in plasma of Gaucher patients, was measured in patients with β-thalassemia, an haematological disorder characterized by the genetic defect of β-globin chains synthesis resulting in unproductive erythropoiesis and enormous expansion of the reticuloendothelial system. Plasma chitotriosidase was increased to a variable extent in 13 of 70 patients with β-thalassemia major treated with the intense transfusion regimen and iron chelation therapy. It was normal in 22 and slightly elevated in 3 subjects with β-thalassemia intermedia which were not transfused. The highest levels of plasma chitotriosidase, as high as in Gaucher patients, were found in 7 (10%) of the β-thalassemia major patients which also had the highest degree of iron overload as judged by their serum ferritin level (> 3000 ng/ml), high SGPT level and elevated urinary iron excretion. To our knowledge, β-thalassemia is hitherto the only disorder in which an increase of plasma chitotriosidase, comparable to that seen in Gaucher disease, may occur. The increase of plasma chitotriosidase activity in β-thalassemia patients with high iron overload, could be related to an iron mediated damage to the lysosomal apparatus. In addition, similarly to Gaucher disease, the increased chitotriosidase production in β-thalassemia might reflect macrophage activation probably related to the intracellular iron overload, storage of erythrocytes membrane break-down products and oxidation of excess alpha-hemoglobin subunits. Further studies are required to define the role of chitotriosidase evaluation to assess the efficacy of chelation therapy in reducing the macrophage activation due to intracellular iron overload in β-thalassemia.

Blood antioxidant status and urinary levels of catecholamine metabolites in β-thalassemia

It has been reported that iron overload in β-thalassemia leads to an enhanced generation of reactive oxygen species and to oxidative stress. We have studied the oxidant/antioxidant imbalance in the blood of 48 transfusion-dependent β-thalassemic patients (TLP) (17 males, 31 females, 11–22 year), under chelation therapy, and in 40 sex and age matched healthy controls (CTR). Plasma and lymphocyte levels of vitamin E (Vit E), ubiquinol (CoQ10H2), ubiquinone (CoQ10), plasma concentrations of vitamin A (Vit A), β-carotene, lycopene, vitamin C (Vit C), total thiols, fatty acid patterns of phospholipids (PL-FA), and plasma and urinary markers of lipoperoxidation (TBA-RM, conjugated dienes, and azelaic acid (AZA), as well as the urinary levels of catecholamine and serotonin metabolites, were evaluated by gas chromatography-mass spectrometry (GC-MS), HPLC and spectrophotometry. Routine laboratory blood analyses were performed on the same samples; 39/48 TLP were HCV positive. Blood samples were collected just before transfusion, the 24 h urine samples the day before. Our results clearly showed that a severe oxidative stress occurs in the plasma of TLP in comparison with CTR. In fact, the levels of lipophilic antioxidants and ascorbate were severely depleted: CoQ10H2 (-62.5%), total CoQ10 (-35.1%), Vit E (-43.8%, β-carotene (-31.1%), lycopene (-63.7%), Vit A (-35.9%), Vit C (-23.1%). The impairment of the antioxidant status was associated with elevated plasma levels of by-products of lipoperoxidation and urinary concentrations of catecholamine metabolites and of AZA, indicating a high degree of both neurological stress and lipoperoxidation. A significant positive correlation was found between vitamin E and non-transferrin-bound iron (NTBI) (r = -0.81; p < 0.001), while no correlation was found between antioxidant depletion and ferritin serum levels, average blood consumption, or the presence of clinical complications. The administration of selective antioxidants along with an appropriate diet might represent a promising way of counteracting oxidative damage and its deleterious effects on the progression of the disease.