Thalassemic Mice Research Articles

Reciprocal correlation between iron and calcium transport and the hepcidin-enhanced calcium absorption in the duodenum of hemizygous beta-globin knockout thalassemic mice

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Dual T-type and L-type calcium channel blocker exerts beneficial effects in attenuating cardiovascular dysfunction in iron-overloaded thalassaemic mice.

What is the central question of this study? Head-to-head comparison of the therapeutic efficacy among commercial iron chelators and a dual T- (TTCC) and L-type calcium channel (LTCC) blocker on cardiac function, mitochondrial function and the protein expression of cardiac iron transporters in thalassaemic mice in iron-overloaded conditions has not been assessed. What is the main finding and its importance? The dual TTCC and LTCC blocker efonidipine could provide broad beneficial effects in the heart, liver, plasma and mitochondria in both wild-type and thalassaemic mice in iron-overloaded conditions. Its beneficial effects are of the same degree as the three commercial iron chelators currently used clinically. It is possible that efonidipine could be an alternative choice in patients unable to take iron chelators for the treatment of iron-overload conditions. Iron chelation therapy is a standard treatment in thalassaemia patients; however, its poor cardioprotective efficacy and serious side-effects are a cause for concern. Previous studies have shown that treatment with L-type calcium channel (LTCC) blockers or dual T-type calcium channel (TTCC) and LTCC blockers decreases cardiac iron and improves cardiac dysfunction in an iron-overloaded rodent model. Currently, the head-to-head comparison of therapeutic efficacy among commercial iron chelators, a dual TTCC and LTCC blocker and an LTCC blocker on cardiac function, mitochondrial function and the protein expression of cardiac iron transporters in thalassaemic mice in an iron-overloaded state has never been investigated. An iron-overloaded state was induced in β-thalassaemic and wild-type mice. Cardiac iron overload was induced to a greater extent than in a previous study by feeding the mice with an iron-enriched diet for 4months. Then, an LTCC blocker (amlodipine) or a dual TTCC and LTCC blocker (efonidipine) or one of the commercial iron chelators (deferoxamine, deferasirox or deferiprone) was administered for 1month with continuous iron feeding. All treatments reduced cardiac iron deposition and improved mitochondrial and cardiac dysfunction in both types of mice. Only efonidipine and the iron chelators reduced liver iron accumulation, liver malondialdehyde and plasma malondialdehyde in these mice. Although all pharmacological interventions reduced cardiac iron deposition, they did not alter the protein expression levels of cardiac iron transporter. These findings indicated that efonidipine provided all benefits to the same degree as the three commercial iron chelators. These findings indicate that a dual TTCC and LTCC blocker could be beneficial for treatment of an iron-overloaded state.

Effects of iron overload condition on liver toxicity and hepcidin/ferroportin expression in thalassemic mice

AimsAlthough iron-overload conditions can be found in β-thalassemic patients, resulting in cellular damage, particularly in the liver, the mechanism for this iron-mediated hepatic injury specifically in β-thalassemic (HT) mice is unclear. This study aimed to investigate the roles of L-type calcium channels (LTCC), T-type calcium channels (TTCC) and divalent metal transporter1 (DMT1) in iron-mediated hepatic injury in HT mice. Main methodsIron chelator deferoxamine (DFO), LTCC blocker, TTCC blocker and DMT1 blocker were used to determine the roles of these channels regarding liver iron accumulation, apoptosis and iron regulatory protein expression in HT mice. Key findingsTTCC and DMT1 blockers and DFO decreased liver iron and malondialdehyde (MDA) in HT mice indicating their antioxidant effects, whereas LTCC blocker produced no decrease in liver iron or MDA. However, only DFO decreased liver apoptosis through the reduced Bax/Bcl-2 ratio in wild type (WT) mice. The levels of iron regulatory hormone hepcidin were markedly higher in HT mice even before iron loading while ferroportin levels did not alter. Each of the pharmacological interventions increased ferroportin protein back to normal levels only in WT while HT mice showed no difference. SignificanceThalassemic mice have different hepcidin/ferroportin and apoptotic protein expression as a defense mechanism to iron-overload compared with those in WT mice. DFO was the most effective intervention in preventing liver apoptosis under iron-overload conditions in WT but did not have the same effect in HT mice.

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.

Alteration of HSC Functions in Thalassemia

Beta-thalassemia represents one of the most globally widespread monogenic disorders and is characterized by significantly reduced or absent synthesis of hemoglobin beta-chains. In its severe form the insufficient production of adult hemoglobin results in altered erythropoiesis, hemolytic anemia, bone marrow (BM) hematopoietic hyperplasia and splenomegaly often associated with extramedullary hematopoiesis, requiring regular blood transfusions and iron chelation treatment. Over the last two decades many progresses were made in the field of allogeneic bone marrow (BM) transplantation to definitively cure beta-thalassemia. In parallel, experimental autologous transplantation protocols were developed to correct the disease by gene therapy also in patients lacking a compatible donor. Both in the allogeneic and autologous setting, thalassemic hematopoietic stem cells (HSCs) and the BM niche represent central elements.Although many aspects of the pathophysiology of thalassemia have been extensively investigated, the HSC and its niche have never been explored. In thalassemia, the BM is a stressed environment, characterized by the compensatory expansion of erythroid progenitors secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations, such as primitive progenitors and/or HSCs, might be affected by such an altered hematopoietic microenvironment is unknown.We investigated the frequency of hematopoietic progenitors in a murine model of severe beta-thalassemia intermedia. Immunophenotypic analyses revealed no differences in MEP, GMP, CMP, LMPP and MPP committed precursor subpopulations, whereas a significantly lower frequency of HSCs (Lin- Sca-1+ c-kit+ CD48- CD150+) was observed in thalassemic mice, as compared to age-matched wild-type controls. Competitive transplantation experiments revealed a disadvantage in the engraftment capacity of thalassemic HSCs, which was substantiated by the preliminary results from in vitro and in vivo cell cycle analyses suggesting an accelerated HSC exhaustion. Analyses of other cellular components, such as BM stroma and differentiated hematopoietic cells, revealed that additional elements are altered in the thalassemic BM microenvironment. The cellular and molecular bases of HSC-niche interaction in this pathological condition are under investigation.Our results uncover a previously ignored defect of HSCs in beta-thalassemia. The investigation of cellular and molecular players that might affect in trans HSC functions in the complexity of this altered microenvironment is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiology and Treatment of Beta-Thalassemia: Investigations of Heme Oxygenase 1 and Its Inhibitors

Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts. "Unshielded" heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme.So far, virtually no information about the expression of HO-1 in erythroblasts has been produced. However, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1-mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3. Our data indicates that HO-1 expression is increased in the liver, spleen and kidney of β-thalassemic mice compared to wild-type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses have increased levels of HO-1 at mRNA and protein levels as well as a decrease in phosphorylated eIF2-α levels. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release. To investigate the contribution of HO-1 to the pathology associated with β-thalassemia, wild-type and thalassemic (th3/+) mice were injected with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO-1 inhibitor) during a 4-week period, 3 times a week. Our results show that β-thalassemic mice injected with SnPP display a decrease in the spleen index, hemoglobin levels, red blood cell counts, reticulocyte counts and liver iron content when compared to PBS injected β-thalassemic mice. Additionally, HO-1 inhibition reduced ineffective erytropoiesis in β-thalassemia mice. Our results indicate that β-thalassemic erythroblasts have inappropriately high levels of "free" heme that is continuously degraded by HO-1. Further research is needed to determine whether iron liberated from heme by HO-1 is directly responsible for the damage of β-thalassemic erythroblasts.1Garcia-Santos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 2269-77, 2014. DisclosuresNo relevant conflicts of interest to declare.

Lack of Beta-1 Integrin Limits Stress Erythropoiesis and Splenomegaly in Beta-Thalassemia

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ1 expression, indicating that Itgβ1 may be involved in erythroblast enucleation and differentiation. We crossed Hbbth3/+ mice with animals in which Itgβ1 or Fak1 were floxed and carrying an inducible Cre−recombinase (Mx1−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbbth3/+−Itgβ1fl/fl−Mx1−Cre and Hbbth3/+−Fak1fl/fl−Mx1−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbbth3/+−Itgβ1fl/fl −Mx1−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ1 only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbbth3/+−Itgβ1fl/fl−Mx1−Cre animals generated through BMT. Therefore, Itgβ1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. DisclosuresFeldman:Bayer ealthCare Phamaceuticals Inc.: Employment. Rivella:isis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; Novartis Pharmaceuticals: Consultancy; Medgenics Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Inactivation of Tmprss6 Hampers Hepcidin Inhibition By Erythroferrone through Upregulation of the BMP-SMAD Pathway

IntroductionErythroferrone (ERFE), a member of the C1q-TNF protein family produced by erythropoietin (EPO)-stimulated erythroid cells, has been recently identified as the erythroid regulator that suppresses hepcidin (Kautz et al., 2014). In the liver, the main hepcidin inhibitor is TMPRSS6, the only known in vivo inhibitor of the BMP-SMAD pathway since its inactivation causes Iron Refractory Iron Deficiency Anemia (IRIDA) (Finberg et al., 2008), a genetic disorder due to inappropriately high hepcidin. We have previously demonstrated that inactivation of Tmprss6 in thalassemia mice rescues iron overload and ameliorate anemia by increasing hepcidin, notwithstanding high Epo (Nai et al., 2012) and Erfe (Silvestri et al., ASH Meeting 2014) thus suggesting that Tmprss6 is indispensable for hepcidin inhibition by the erythroid regulator(s).MethodsTo analyze BM and spleen erythroid differentiation, Ter119+ cells were separated according to Forward Scatter (FSC) value and Cd44 expression, as previously described (Liu et al., 2013). Expression of Erfe, Epo, hepcidin and BMP-SMAD target genes were evaluated by qRT-PCR and iron parameters by standard methods (Pagani et al., 2011). We have analyzed different animal models after a single (200U) EPO injection: 1) Tmprss6 KO mice and wild type littermates fed an iron balanced (IB) and iron deficient (ID), diet; 2) wild type mice with activated BMP-SMAD pathway by iron dextran (1g/kg) treatment; 3) Tmprss6 KO animals with the BMP-SMAD pathway inhibited by 12.5 μg/g dorsomorphin. Both males and females mice were analyzed at 8-10 weeks of age and sacrificed 15 hours after EPO treatment.ResultsEPO administration increases bone marrow (BM) erythroid precursors in all mice models. In the spleen erythroid precursors are not further upregulated by EPO in ID and Tmprss6 KO mice, since already high in basal conditions likely due to stress erythropoiesis. Erfe expression is upregulated in basal conditions in BM and spleen of ID and Tmprss6 KO mice, which have high serum Epo levels, and is further increased by EPO treatment in all models. As expected, hepcidin is strongly reduced in ID animals, whereas it is high in Tmprss6 KO mice. EPO injection strongly inhibits hepcidin both in IB and ID mice but not in Tmprss6 KO animals, notwithstanding the upregulation of Erfe. This suggests that lack of Tmprss6 impairs the Erfe-mediated hepcidin inhibition. To evaluate the contribution of Tmprss6 and the BMP signaling to Erfe function, we modulated the BMP pathway before EPO challenge. We activated the signaling by means of iron dextran injection in wild type mice and we inhibited the hyperactive pathway of Tmprss6 KO animals by using the BMP receptor inhibitor dorsomorphin. Neither iron dextran injection nor dorsomorphin treatment impairs Erfe activation by EPO that equals that observed in saline treated animals. In iron loaded mice with hyperactive BMP signaling, hepcidin was not inhibited by Erfe. Dorsomorphin treatment in Tmprss6 KO animals rescues the hepcidin responsiveness to Erfe.ConclusionsWe confirm that the BMP-SMAD pathway is dispensable for Erfe-mediated hepcidin inhibition since Erfe decreases hepcidin in ID, when the pathway is suppressed. However, activation of the BMP-SMAD pathway consequent to iron loading or to the loss of Tmprss6 hampers the hepcidin downregulation by Erfe, suggesting that the Tmprss6 function, and not Tmprss6 per se, is essential to facilitate the Erfe-dependent hepcidin inhibition by blunting the BMP-SMAD signaling. DisclosuresNo relevant conflicts of interest to declare.

Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of β-thalassemia

AbstractInherited anemias with ineffective erythropoiesis, such as β-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron, leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of HbbTh3/+ mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age, suggesting ERFE could be a factor suppressing hepcidin production in β-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age, and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis, as indicated by reduced spleen index, red cell distribution width, and mean corpuscular volume, but did not improve the anemia. Thus, ERFE mediates hepcidin suppression and contributes to iron overload in a mouse model of β-thalassemia.

Bone microstructural defects and osteopenia in hemizygous βIVSII-654 knockin thalassemic mice: sex-dependent changes in bone density and osteoclast function.

β-Thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both males and females. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in β(IVSII-654) knockin thalassemic mice. Dual-energy X-ray absorptiometry and computer-assisted bone histomorphometry were employed to investigate temporal changes in BMD and histomorphometric parameters in male and female mice of a β(IVSII-654) knockin mouse model of human β-thalassemia, in which impaired splicing of β-globin transcript was caused by hemizygous C→T mutation at nucleotide 654 of intron 2. Young, growing β(IVSII-654) mice (1 mo old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 mo of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between sex and β(IVSII-654) genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male β(IVSII-654) mice, particularly during a growing period (1-2 mo), was primarily due to suppression of bone formation, whereas both a low bone formation rate and a marked increase in osteoclast surface were observed in female β(IVSII-654) mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female β(IVSII-654) knockin thalassemic mice, but the severity, disease progression, and cellular mechanism differed between the sexes.

Combined treatment of 3-hydroxypyridine-4-one derivatives and green tea extract to induce hepcidin expression in iron-overloaded β-thalassemic mice

ObjectiveTo evaluate the efficacy of deferiprone (DFP), 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) or green tea extract (GTE) in enhancing expression of hepatic hepcidin1 (Hamp1) mRNA and relieving iron overload in β-globin knockout thalassemic mice. MethodsThe β-globin knockout thalassemic mice were fed with a ferrocene-supplemented diet for 2 months and oral administration of deionized water, DFP (50 mg/kg), CM1 (50 mg/kg), GTE (50 mg epigallocatechin 3-gallate equivalent/kg), GTE along with DFP (50 mg/kg), and GTE along with CM1 (50 mg/kg) every day for 3 months. Levels of hepatic Hamp1 mRNA, plasma non-transferrin bound iron, plasma alanine aminotransferase activity and tissue iron content were determined. ResultsAll chelation treatments could reduce plasma non-transferrin bound iron concentrations. Additionally, hepatic Hamp1 mRNA expression was significantly up-regulated in the mice in a GTE + DFP combined treatment, correlating with a decrease in the plasma alanine aminotransferase activity and tissue iron deposition. ConclusionsThe GTE + DFP treatment could ameliorate iron overload and liver oxidative damage in non-transfusion dependent β-thalassemic mice, by chelating toxic iron in plasma and tissues, and increasing hepcidin expression to inhibit duodenal iron absorption and iron release from hepatocytes and macrophages in the spleen. There is probably an advantage in giving GTE with DFP when treating patients with iron overload.

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

A mouse model to study thrombotic complications of thalassemia

Patients with β-thalassemia major and mainly intermedia have an increased risk for developing venous and arterial thrombosis which may be related to circulating pathological red blood cells (RBC) and continuous platelet activation. In the present study we used a modified thalassemic mice model in conjunction with a "real-time" carotid thrombus formation procedure to investigate thrombotic complications of thalassemia. Heterozygous Th3/+ mice, which lack one copy of their β-major and β-minor globin genes, exhibit anomalies in RBC size and shape, chronic anemia and splenomegaly which recapitulate the phenotype of human β-thalassemia intermedia. Flow cytometry measurements showed higher reactive oxygen species generation, indicating oxidative stress, in platelets and RBC of the thalassemic mice compared with wild type mice concomitant with an increase in reduced glutathione content which may represent a compensatory response to oxidative stress, and exposed phosphatidylserine which indicates platelet activation. To elucidate the effect of thalassemia on the development of arterial thrombosis, we studied photochemical-induced real-time thrombus formation in the carotid artery of these mice. The results indicated a significantly shorter "time to occlusion" in the thalassemic mice compared to wild type mice, which was prolonged following in vivo aspirin treatment. We suggest that this mouse model may contribute to our understanding of platelet activation and the hypercoagulable state in thalassemia and lay foundations to screening of anti-platelet drugs as well as anti-oxidants as possible therapeutics for prevention of thrombosis in thalassemia patients.

Construction of thalassemic mouse induced pluripotent stem cells for disease modeling

Induced pluripotent stem cells (iPSCs) have a great potential for disease modeling and therapeutic applications. One of the most common genetic diseases found in South East Asia is thalassemia. β-Thalassemia is an inherited single gene disorder caused by reduction or absence of β-globin chain production. The purpose of this study is to generate mouse iPSCs with β-thalassemia background. Mouse embryonic fibroblast (MEF) cells were obtained from transgenic β-thalassemic mouse, transduced with lentiviral vector carrying 4 transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), and cultured on a feeder layer. The embryonic stem (ES)-like colonies were manually picked up and expanded. The generated mouse iPSCs were similar to mouse embryonic stem cells (mESCs) in morphology, alkaline phosphatase activity and pattern of pluripotent gene expression and able to form teratoma containing tissues derived from all three germ layers. Therefore, we reported here an establishment of β-thalassemic mouse iPSCs which might be used for further disease modeling and therapeutic applications.

Increased Hepcidin Expression in β-Thalassemic Mice Treated with Apo-Transferrin Is Associated with Increased Smad1/5/8 and Decreased Erk1/2 Pathway Activation

Iron overload causes morbidity and mortality in patients with β-thalassemia. Transfusion independent patients develop iron overload from increased dietary iron absorption, implicating inappropriately low hepcidin and supporting the therapeutic potential of approaches to increase hepcidin. Relatively low liver hepcidin mRNA expression is also characteristic of mouse models of β-thalassemia, and a recently identified “erythroid factor,” erythroferrone, has been implicated in hepcidin suppression in thalassemia. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoiesis, ameliorate ineffective erythropoiesis, and increase hepcidin expression in Hbbth1/th1 β-thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and in vitro in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin injection increase both serum hepcidin concentration (603 vs. 306 ng/ml, n=13-24 per group, P<0.0001) and liver mRNA expression (2.3-fold, n=8-12 per group, P=0.003) despite decreased circulating serum iron (96 vs. 180 μg/ml, n=6-8 per group, P=0.001) and parenchymal liver iron (1.0 vs. 1.3 µg/mg, n=10-12 per group, P=0.04) concentrations. Increased hepcidin in apo-transferrin treated thalassemic mice is unrelated to changes in liver Bmp6 mRNA expression (1.1-fold increase, n=8 per group, P=0.49) but correlates well with serum BMP2 concentration (1.3-fold increase, n=6-9 per group, P=0.03). Freshly isolated hepatocytes from thalassemic mice exhibit more pErk1/2 and a higher ratio of pErk1/2:Erk1/2 relative to WT mice, and apo-transferrin treated thalassemic mice exhibit a significant suppression of the Erk1/2 pathway (Figure 1) and increased Smad activation (1.4-fold increase, n=4-6 per group, P=0.02). These findings strongly suggest an inhibitory effect of the Erk pathway on hepcidin expression. We thus evaluate the effect of Erk inhibitor U0126 on freshly isolated hepatocytes from WT mice and demonstrate that erk inhibition in vitro also results in a dose-dependent increase in hepcidin expression (3.7-fold increase at 50µM U0126, P=0.0003) with no change in Smad1/5/8 phosphorylation. To further evaluate the role of circulating factors on the regulation of hepcidin expression with apo-transferrin treatment, we analyzed the effect of serum from PBS- and apo-transferrin treated WT and thalassemic mice on cultured primary hepatocytes from WT mice. A significant increase in hepcidin expression is observed in cells exposed to serum relative to untreated cells (Figure 2), while hepatocytes treated with serum from thalassemic mice demonstrate suppressed hepcidin expression and pSmad1/5/8 relative to WT serum (Figure 2 and 3). Furthermore, primary hepatocytes concurrently treated with serum and neutralizing anti-BMP2/4 antibodies have relatively suppressed hepcidin expression in each condition relative to cells treated with serum alone (Figure 2), suggesting again the importance of BMP2 in hepcidin expression. The treatment with serum and neutralizing anti-BMP2/4 antibodies also suppressed Smad1/5/8 and induces Erk1/2 pathway activation (Figure 3). Lastly, erythroferrone expression is increased in sorted orthochromatophilic bone marrow erythroblasts in thalassemic relative to WT mice and normalized by apo-transferrin injection in thalassemic mice (2.4-fold increase thalassemic vs. WT mice (P=0.008); 2-fold decrease apoTf-treated vs. PBS-treated thalassemic mice (P=0.03)). No differences are observed either in GDF15 or TWSG1 in sorted bone marrow orthochromatophilic erythroblasts. These findings support the importance of erythroferrone as an erythroid regulator in thalassemic mice, suggest that the effect in Hbbth1/th1 mice and Hbbth3/+ mice are comparable, and provides further evidence that treatment with apo-transferrin reverses ineffective erythropoiesis in thalassemic mice. In total, our findings support a model in which treatment of Hbbth1/th1 mice with apo-transferrin decreases bone marrow erythroferrone expression, decreases hepatocellular Erk activation, and increases Smad activation to increase liver hepcidin expression. [Display omitted] [Display omitted] [Display omitted] DisclosuresWesterman:Intrinsic Lifesciences LTD: Employment, Equity Ownership.

Beta-1 Integrin Controls Homing and Expansion of Erythroid Cells in Stress Erythropoiesis and ß-Thalassemia

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β-thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes.Interupting the interaction between macrophages and erythroblasts (macrophage-erythroblast interaction, MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. Targeting MEI, using a number of approaches, caused a significant improvement in blood parameters in β-thalassemia intermedia (BTI) mouse models (Hbbth3/+) and a rapid and dramatic improvement in splenomegaly, an outcome that is relevant for clinical practice. Importantly, MEI is not critical for hematopoiesis under non-stress conditions, and ablation of this interaction in normal mice showed minimal effects on blood parameters. As our initial observations indicate that MEI is essential to support stress erythropoiesis, we investigated adhesion molecules that might activate downstream pathways in erythroblasts that regulate cell proliferation. We also speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver.Our studies in erythroblasts indicate that integrin beta 1 (Itgb1) and also intracellular molecules such as Fak1, Talin1 and Sharpin might play a role in stress erythropoiesis. There is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgb1 and Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgb1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. More specifically, when measuring the content of DNA, we were able to demonstrate that enucleation of erythroblasts was accompanied by a marked loss of Itgb1 expression, indicating that there may be an important role for Itgb1 in erythroblast enucleation, and differentiation in general. Lack of Itgb1 in thalassemic mice prevents erythroid cells from homing to and expanding in the spleen, the major source of chronic stress erythopoiesis in this disorder. In particular, such a role of Itgb1 is supported by our analysis of thalassemic mice in which this molecule was partially depleted by induction of the Cre recombinase. These animals were generated by crossing th3/+ mice with animals in which Itgb1 was floxed and carrying an inducible Cre-recombinase (Mx1-CRE). We utilized the BM of these animals (Hbbth3/+, Itgb1fl/fl, Mx1-CRE) to generate thalassemic animals that expressed the floxed Itgb1 only in hematopietic cells. After serial administration of polyI:C the animals were analyzed for their erythropoiesis in the bone marrow and spleen. Interestingly, all the animals analyzed show chimeric populations of Itgb1 positive and negative erythroid cells in the bone marrow. This indicated that not all the HSCs were successfully depleted of the Itgb1 gene. However, when we investigated Itgb1 in the spleen, we observed only erythroid cells positive for the expression of this adhesion molecule. This last observation strongly suggests that depletion of Itgb1 prevents homing and expansion of erythroid cells in the spleen and drugs that by inhibit Itgb1 could reduce erythroid spleen colonization, splenomegaly and limit erythropoiesis.We are now in the process of identifying compounds that target MEI. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. DisclosuresCasu:Merganser Biotech : Research Funding; Isis Pharmaceuticals, Inc.: Research Funding.

Ineffective Erythropoiesis Is the Major Cause of Microcytic Anemia in the TSAP6/Steap3 Null Mouse Model

STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p<0.001) and mean cell hemoglobin content (MCH; 5.8pg ± 0.1pg vs 14.5pg ± 0.1pg, p<0.001). The reticulocyte count was marginally elevated (5.7% ± 1.2% vs 2.6% ± 0.4%, p<0.05) indicating that the anemia is proliferative. By phase contrast microscopy and transmission electron microscopy, we observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with this observation, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. Interestingly, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability.As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p<0.01). In addition, progression from the proerythroblastic to the orthochromatic stage was also affected, with accumulation of cells at the polychromatic stage. These findings imply that ineffective erythropoiesis is the dominant cause of anemia in these mice and that TSAP6/Steap3 may play a role during erythropoiesis beyond its role as ferrireductase. It is interesting to note that while the phenotype of microcytic red cells with decreased hemoglobin content in the TSAP6/Steap3 null mouse is similar to red cells in thalassemic and porphyria mice, there are distinct differences in the etiology of the hematologic phenotype. DisclosuresNo relevant conflicts of interest to declare.

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.

Iron Overload in the Face of Anemia

Several distinct congenital disorders can lead to tissue-iron overload with anemia including β-thalassemia and sickle cell disease. We show that intestinal absorption of iron is highly increased and significantly contributes to tissue iron accumulation in these disorders. The present work describes a novel pathway by which oxygen sensing transcription factors are highly upregulated in iron overload anemias and are subsequently essential for the increase intestinal iron absorption. Oxygen signaling is mediated through well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)1a and HIF2a. In the intestine, HIF2a directly activates divalent metal transporter 1 (DMT1), duodenal ferric reductase (DcytB), and Fpn1, which are iron transporters critical for adaptive changes in iron absorption. We demonstrate that HIF2a and its downstream target gene, DMT1 are essential for iron accumulation in mouse models of β-thalassemia and sickle cell disease. Furthermore, studies of thalassemic mouse model with established iron overload demonstrated that loss of intestinal HIF2a and DMT1 signaling led to decreased tissue iron accumulation in the livers. Interestingly, disrupting intestinal HIF2a not only improves tissue iron accumulation, but a marked improvement of anemia was also observed. These novel findings suggests that inhibition of HIF2a signaling pathway could be a novel and robust treatment strategy for several conditions that cause iron overload with anemia. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic effects of induced pluripotent stem cells in chimeric mice with β-thalassemia.

Although β-thalassemia is one of the most common human genetic diseases, there is still no effective treatment other than bone marrow transplantation. Induced pluripotent stem cells have been considered good candidates for the future repair or replacement of malfunctioning organs. As a basis for developing transgenic induced pluripotent stem cell therapies for thalassemia, β(654) induced pluripotent stem cells from a β(654) -thalassemia mouse transduced with the normal human β-globin gene, and the induced pluripotent stem cells with an erythroid-expressing reporter GFP were used to produce chimeric mice. Using these chimera models, we investigated changes in various pathological indices including hematologic parameters and tissue pathology. Our data showed that when the chimerism of β(654) induced pluripotent stem cells with the normal human β-globin gene in β(654) mice is over 30%, the pathology of anemia appeared to be reversed, while chimerism ranging from 8% to 16% provided little improvement in the typical β-thalassemia phenotype. Effective alleviation of thalassemia-related phenotypes was observed when chimerism with the induced pluripotent stem cells owning the erythroid-expressing reporter GFP in β(654) mouse was greater than 10%. Thus, 10% or more expression of the exogenous normal β-globin gene reduces the degree of anemia in our β-thalassemia mouse model, whereas treatment with β(654) induced pluripotent stem cells which had the normal human β-globin gene had stable therapeutic effects but in a more dose-dependent manner.