Hepcidin Suppression Research Articles

High levels of soluble serum hemojuvelin in patients with congenital dyserythropoietic anemia type I

Congenital dyserythropoietic anemia (CDA) is a rare group of red blood cell disorders with ineffective erythropoiesis and secondary hemochromatosis. Inappropriate suppression of hepcidin and high levels of growth differentiation factor 15 (GDF15) have been described in CDA I and II patients, probably contributing to secondary hemochromatosis. Hemojuvelin (HJV) is an important regulator of serum hepcidin, while soluble form of HJV (s-HJV) competitively down-regulates hepcidin. We determined the soluble hemojuvelin (s-HJV) levels in 17 patients with CDA I and in 17 healthy volunteers (HV) and looked for correlations with other parameters of iron overload and erythropoiesis. Significantly higher levels of s-HJV were found in patients (2.32±1.40mg/L) compared with healthy volunteers (0. 69±0.44mg/L) (P=0.001). Western blot analysis confirmed the presence of high levels of s-HJV in CDA I patients. s-HJV positively correlated with serum ferritin, erythropoietin, soluble transferrin receptor, and GDF15 and negatively correlated with hepcidin to ferritin ratios. We for the first time documented high levels of serum s-HJV in CDA I patients, suggesting that it may contribute to iron loading pathology in CDA I and eventually in other anemias with ineffective erythropoiesis.

Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Abstract 481Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia.Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to <150ug/dl in treatment group), as well as liver iron accumulation. Histopathological evaluation and Prussian’s Perl Blue staining indicated that iron was sequestered by macrophages, which led to an increase in spleen iron concentration.The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes.In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures:Guo:Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

Single and Repeated Dose First-in-Human Study with the Anti-Hepcidin Spiegelmer Nox-H94.

AbstractAbstract 2342 Background:NOX-H94, the first-in-class hepcidin inhibitor in development for treatment of anemia of chronic disease (ACD), is a PEGylated anti-hepcidin L-RNA oligonucleotide. ACD is caused by iron sequestration in the reticulo-endothelial macrophages with subsequent iron restricted erythropoiesis due to high hepcidin production and subsequent ferroportin degradation. The treatment of ACD is challenging: a significant number of ACD patients do not respond to erythropoiesis stimulating agents (ESAs), while repeated intravenous iron administrations bear a risk of iron overload. Targeting hepcidin may provide more efficacious and well tolerated treatment alternatives. Methods:This First-in-Human study investigated the safety and tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of escalating single and repeated doses of intravenous (i.v.) NOX-H94 in healthy men and women. The study protocol (ClinicalTrials.gov identifier NCT01372137) was approved by an independent ethics committee and conducted in accordance with the Declaration of Helsinki. Five successive cohorts of 8 healthy subjects, at least 3 men and women, were randomly assigned to i.v. doses of 0.3, 0.6, 1.2, 2.4, and 4.8 mg/kg of NOX-H94 (n=6) or placebo (n=2). Similarly, 2 cohorts of 8 male subjects randomly received 5 doses of either 0.6 or 1.2 mg/kg NOX-H94 or placebo every other day (q2d). Safety parameters, iron parameters, total hepcidin-25 (sum of free circulating hepcidin-25 and hepcidin-25 bound to NOX-H94), and PK were assessed during treatment and follow-up periods of ≥3 weeks. Data are given as means±SD. Results:One man, assigned to 5 i.v. doses of 0.6 mg/kg, withdrew consent after 4 administrations; all other 55 subjects completed the study as scheduled. Safety: Treatment with NOX-H94 was generally safe and well tolerated. No serious adverse event occurred; headache and fatigue were the only treatment related events that occurred more than once. Mild and transient increases in transaminases (<2×ULN) were noted in subjects treated with NOX-H94 at single doses ≥2.4 mg/kg or with repeated doses of 1.2 mg/kg (4.2 mg/kg weekly). PK: After escalating single i.v. administrations of 0.3 to 4.8 mg/kg of NOX-H94, peak plasma concentrations of NOX-H94 (Cmax) and systemic exposure (AUC) increased dose-proportionally. The elimination was bi-phasic with a terminal plasma half-life (t ½) in the range of 17 to 26 h. The systemic clearance (CL) was low (Table 1). After repeated q2d i.v. administrations, no appreciable plasma accumulation was found based on Cmax and AUC. No obvious gender-difference was observed. PD: The plasma concentration of total hepcidin-25 increased dose-dependently upon NOX-H94 treatment, without ever exceeding the plasma concentration of NOX-H94. The PD effects were assessed by analysis of the area under the data time curve above baseline (AUD) of various iron parameters. Single and repeated doses of NOX-H94 up to 0.6 mg/kg had no effect on serum iron, serum ferritin, and transferrin saturation (TSAT) in the healthy subjects studied. At doses ≥1.2 mg/kg NOX-H94, serum iron, serum ferritin, and TSAT increased dose dependently.Table 1:Single dose PK of NOX-H94Dose Group [mg/kg]nCmax [μM]AUC 0-tlast [μM×h]t ½ [h]CL [L/h]0.360.524±0.0985.63±3.4316.5±7.50.34±0.220.661.09±0.36013.0±4.5518.0±7.50.24±0.101.262.17±0.26130.4±6.2423.9±9.90.18±0.032.464.75±0.67980.0±12.023.5±4.40.15±0.034.869.85±1.53213±26.526.3±3.20.12±0.03Table 2:Serum iron parameters after single dose treatment with NOX-H94 analyzed based on the area under the data time curve above baseline (means±SD)Dose Group [mg/kg]nSerum iron [μmol/L×h]Serum ferritin [μg/L×h]TSAT [%×h]Placebo1044.6±28.429.5±45.362.5±41.00.3647.6±51.915.5±31.382.2±76.90.6667.0±40.344.4±53.2111±60.11.26193±53.7125±205321±1372.46134±45.0371±290239±97.84.86225±138644±670422±334 Conclusions:Treatment with NOX-H94 was generally safe and well tolerated over all dose levels and schedules studied; for subsequent phase II studies in patients, twice weekly i.v. doses of 1.2 mg/kg are recommended. PK analyses showed a dose-linear systemic exposure. In these healthy subjects, only mild dose-dependent increases in iron parameters were observed which likely underestimate the effects that may be obtained in patients with iron-restricted anemia. Disclosures:Riecke:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Vauléon:NOXXON Pharma AG: Employment. Swinkels:NOXXON Pharma AG: Research Funding. Dümmler:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

Hepcidin Inhibition by Modified Heparins without Anticoagulant Activity

AbstractAbstract 483 Background.There is an increasing interest in the development of pharmacological agents able to modulate hepcidin, the peptide hormone that critically regulates iron metabolism. In particular, hepcidin antagonist may have a therapeutic role in the anemia of chronic diseases, where hepcidin levels are often increased by pro-inflammatory cytokines. We previously demonstrated that heparin is a potent inhibitor of hepcidin expression in hepatic cell lines, probably by interfering with BMP/HJV/SMAD signalling, and that it was also effective in reducing hepcidin expression in mice (Poli M, Blood 2011; 117:997–1004). Since the therapeutic use of heparin for hepcidin modulation is hampered by its strong anticoagulant activity, we were interested in evaluating modified heparins without such activity. Methods.Heparins modified to inactivate the antithrombin binding site, with different molecular weight and degree of sulfation, were supplied to hepatic cell lines and mice to evaluate their potential modulation of hepcidin expression. We analysed their interference with the BMP/SMAD signalling, as well as serum hepcidin levels in mice by mass spectrometry. Results.Over 20 modified heparins were initially screened by evaluating their dose-dependent suppression of hepcidin expression before and after BMP induction. All of them showed a certain degree of anti-hepcidin activity, with two glycol-split molecules being as potent as classical unfractionated heparin. These two molecules suppressed BMP/SMAD signalling in HepG2 cells at pharmacological concentrations with maximum inhibition after 6 hours. In mice, treatments with 20 or 60 mg/Kg did not affect coagulation but strongly reduced liver pSMAD, hepcidin mRNA and serum hepcidin. Again, the maximum effect on liver hepcidin expression was observed 6 hours after the injection. This effect was observed also in conditions of high hepcidin caused by experimental inflammation or iron overload. Conclusions.Some non-anticoagulant heparins have strong anti-hepcidin activity both in vitro and in vivo, and may represent promising hepcidin antagonist with potential therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

The Red Cell: How Erythropoiesis Modulates Hepcidin Expression

Abstract SCI-24Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders. Disclosures:No relevant conflicts of interest to declare.

High Expression of p53 and Growth Differentiation Factor-15 in Beta-Thalassemia.

Abstract 2130Beta thalassemia phenotype is exacerbated by marrow expansion, tissue hypoxia, and erythroblast apoptosis caused by globin chain imbalances. The expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression. It was shown that growth differentiation factor 15 (GDF15), a member of the transforming growth factor-b super family was elevated in b thalassemia and contributes to hepcidin suppression. p53 plays a key role in the cellular response to stress by activating a subset of genes responsible for cell cycle arrest and apoptosis in tumours. Recently association of P53 with defective erythropoiesis has been demonstrated in Diamond Blackfan anemia. In this study we analyzed the expression of these genes in patients with b thalassemia. We observed a higher expression of p53 and p21 in b thalassemia. Thirty six patients with b thalassemia (b thalassemia major=27; b thalassemia intermedia=9) were included in the study. Diagnosis was based on clinical, haematological and molecular parameters. Healthy volunteers were included as controls (n=28). Serum GDF15 levels were measured using ELISA (Ray Biotech, Inc, Georgia) and ferritin by chemiluminescent immunoassay. Reticulocytes from patients and controls were isolated from peripheral blood using cellulose columns. RNA was extracted by Trizol method and cDNA was synthesised. The relative expression of p53, GDF15 and p21 mRNA was analyzed using actin as the control gene. Approximately 1×107 reticulocytes were used to prepare whole cell lysate for western blot (controls=6, patients=6). As expected serum ferritin (Median-3544ng/ml) and GDF15 levels (Median-6059.87pg/ml) were higher in the patient cohort as compared to controls. Higher expression of p21 and p53 was observed in patients as compared to controls (Table.1). P53 expression strongly correlated with GDF15 expression at the mRNA (r=0.649; p=0.000) and protein level (r=0.406; p=0.049) in b thalassemia major. The p53 expression also significantly correlated with serum ferritin levels (r=0.562; p=0.003) in the thalassemia major cohort. We also assessed the expression of p21 one of the targets of p53 in these samples. Significantly higher expression of p21 was observed in the patients than in controls (Median dCt-8.34 vs 10.78; p=0.000). Western blot analysis carried out in six b-thalassemia major and six controls also showed a higher expression of p53 in the patients as compared to controls. Higher expression of these genes was also observed in b thalassemia intermedia as compared to controls but not different from thalassemia major (Table.1). GDF15 has two p53 response elements in the promoter region and p53 binding activates GDF15 in the erythroid compartment (Osada et.al 2007). P53 may thus contribute to GDF15 elevation in beta thalassemia.Table 1Gene expression in beta thalassemiaFerritin (ng/ml)GDF15 (pg/mL)Gene Expression Median dCt (Range)GDF15p53p21b Thalassemia Major (n=27)3544 (569-12631)6059.87 (2518-29379)2.97 (−5.28-8.81)7.68 (ϕ1.75-10.48)8.33 (4.87-10.9)b Thalassemia Intermedia (n=9)950.35 (84.80-6634)7576.52 (5391-16014)4.27 (−3.08-8.77)7.69 (−7.41-10.91)8.03 (6.03-10.82)Controls (n=28)26.25 (5-276)328.95 (125-677)5.92 (0.88-10.93)10.15 (4.68-12.84)10.78 (5.65-13.80) Disclosures:Edison:Department Of Biotechnology, Government of India: Research Funding.

A Hepcidin Inhibitor Mobilizes Iron for Incorporation Into Red Blood Cells in an Adenine-Induced Chronic Kidney Disease Model in Rats.

AbstractAbstract 2082 Background:Anemia is prevalent in patients with chronic kidney disease (CKD). A key feature of the anemia of CKD is limited iron availability for efficient erythropoiesis despite adequate body iron stores. It is now well established that excess levels of the iron regulatory hormone hepcidin in CKD are responsible for decreasing expression of the iron exporter ferroportin, thereby blocking iron absorption from the diet and iron release from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in patients. Methods:We developed a modified adenine-induced kidney disease model of anemia in rats by giving a diet supplemented with 0.75% adenine for 3 weeks followed by a normal diet for 3 weeks. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which has previously been shown to lower hepcidin levels, was able to mobilize iron into the plasma and improve iron-restricted erythropoiesis in adenine-treated rats. Results:The modified adenine model had a higher survival rate than previously reported models, while maintaining irreversible renal failure and anemia. Adenine-treated rats had increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin, and inappropriately low EPO levels relative to the degree of anemia. LDN-193189 lowered hepatic hepcidin mRNA and mobilized stored iron into plasma in adenine-treated rats. Moreover, the iron was efficiently incorporated into hemoglobin in reticulocytes. However, LDN-193189 alone did not prevent anemia progression in our model. Conclusions:Lowering hepcidin improved iron availability, but did not improve anemia in an adenine-induced kidney disease model in rats. Co-administration of hepcidin lowering agents with erythropoiesis stimulating agents (ESAs) may be useful as a combination therapy to correct iron balance and thereby reduce the ESA dose needed to achieve target hemoglobin levels. Disclosures:Sun:FerruMax Pharmaceuticals Inc: Consultancy. Lin:Ferrumax Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties. Babitt:Ferrumax Pharmaceuticals, Inc: Equity Ownership.

A Hepcidin Inhibitor Mobilizes Iron for Incorporation Into Red Blood Cells in an Adenine-Induced Chronic Kidney Disease Model in Rats.

Abstract 2082 Background: Anemia is prevalent in patients with chronic kidney disease (CKD). A key feature of the anemia of CKD is limited iron availability for efficient erythropoiesis despite adequate body iron stores. It is now well established that excess levels of the iron regulatory hormone hepcidin in CKD are responsible for decreasing expression of the iron exporter ferroportin, thereby blocking iron absorption from the diet and iron release from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in patients. Methods: We developed a modified adenine-induced kidney disease model of anemia in rats by giving a diet supplemented with 0.75% adenine for 3 weeks followed by a normal diet for 3 weeks. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which has previously been shown to lower hepcidin levels, was able to mobilize iron into the plasma and improve iron-restricted erythropoiesis in adenine-treated rats. Results: The modified adenine model had a higher survival rate than previously reported models, while maintaining irreversible renal failure and anemia. Adenine-treated rats had increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin, and inappropriately low EPO levels relative to the degree of anemia. LDN-193189 lowered hepatic hepcidin mRNA and mobilized stored iron into plasma in adenine-treated rats. Moreover, the iron was efficiently incorporated into hemoglobin in reticulocytes. However, LDN-193189 alone did not prevent anemia progression in our model. Conclusions: Lowering hepcidin improved iron availability, but did not improve anemia in an adenine-induced kidney disease model in rats. Co-administration of hepcidin lowering agents with erythropoiesis stimulating agents (ESAs) may be useful as a combination therapy to correct iron balance and thereby reduce the ESA dose needed to achieve target hemoglobin levels. Disclosures: Sun:FerruMax Pharmaceuticals Inc: Consultancy. Lin:Ferrumax Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties. Babitt:Ferrumax Pharmaceuticals, Inc: Equity Ownership.

Hypoxia-inducible factor regulates hepcidin via erythropoietin-induced erythropoiesis

Iron demand in bone marrow increases when erythropoiesis is stimulated by hypoxia via increased erythropoietin (EPO) synthesis in kidney and liver. Hepcidin, a small polypeptide produced by hepatocytes, plays a central role in regulating iron uptake by promoting internalization and degradation of ferroportin, the only known cellular iron exporter. Hypoxia suppresses hepcidin, thereby enhancing intestinal iron uptake and release from internal stores. While HIF, a central mediator of cellular adaptation to hypoxia, directly regulates renal and hepatic EPO synthesis under hypoxia, the molecular basis of hypoxia/HIF-mediated hepcidin suppression in the liver remains unclear. Here, we used a genetic approach to disengage HIF activation from EPO synthesis and found that HIF-mediated suppression of the hepcidin gene (Hamp1) required EPO induction. EPO induction was associated with increased erythropoietic activity and elevated serum levels of growth differentiation factor 15. When erythropoiesis was inhibited pharmacologically, Hamp1 was no longer suppressed despite profound elevations in serum EPO, indicating that EPO by itself is not directly involved in Hamp1 regulation. Taken together, we provide in vivo evidence that Hamp1 suppression by the HIF pathway occurs indirectly through stimulation of EPO-induced erythropoiesis.

The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

Inhibition of hepcidin transcription by growth factors

The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its storage, and its systemic distribution. Hepcidin production is often insufficient in chronic hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumulation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mediate hepatic regeneration after liver injury. We examined the effect of these growth factors on hepcidin synthesis by hepatocytes. HGF and EGF treatment of primary mouse hepatocytes, as well as EGF administration in mice, suppressed hepcidin messenger RNA (mRNA) synthesis. The suppression of hepcidin by these growth factors was transcriptional, and was mediated by a direct effect of HGF and EGF on the bone morphogenetic protein (BMP) pathway regulating hepcidin synthesis. We further show that growth factors interfered with nuclear localization of activated sons of mothers against decapentaplegic (Smad) and increased the nuclear pool of the BMP transcriptional corepressor TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors, inhibitors in the PI3 kinase pathway and in the mitogen-activated ERK kinase/extracellular signal-regulated kinase (MEK/ERK) pathway prevented HGF suppression of hepcidin in primary mouse hepatocytes. HGF and EGF suppress hepatic hepcidin synthesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating the nuclear localization of BMP pathway transcriptional regulators including activated Smads1/5/8 and the corepressor TGIF. EGF, HGF, and possibly other growth factors that activate similar pathways may contribute to hepcidin suppression in chronic liver diseases, promote iron accumulation in the liver, and exacerbate the destructive disease processes.

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.

17β-Estradiol Inhibits Iron Hormone Hepcidin Through an Estrogen Responsive Element Half-Site

Interaction of estrogen with iron at the systemic level is long suspected, but direct evidence linking the two is limited. In the present study, we examined the effects of 17β-estradiol (E2) on hepcidin, a key negative regulator of iron absorption from the liver. We found that transcription of hepcidin was suppressed by E2 treatment in human liver HuH7 and HepG2 cells, and this down-regulation was blocked by E2 antagonist ICI 182780. Chromatin immunoprecipitation, deletion, and EMSA detected a functional estrogen responsive element half-site that is located between -2474 and -2462 upstream from the start of transcription of the hepcidin gene. After cloning the human hepcidin promoter into the pGL3Luc-Reporter vector, luciferase activity was also down-regulated by E2 treatment in HepG2 cells. E2 reduced hepcidin mRNA in wild-type mice as well as in hemochromatosis Fe gene knockout mice. In summary, our data suggest that hepcidin inhibition by E2 is to increase iron uptake, a mechanism to compensate iron loss during menstruation. This mechanism may also contribute to increased iron stores in oral contraceptive users.

Iron overload in non-transfusion-dependent thalassemia: a clinical perspective

Iron overload due to increased intestinal iron absorption represents an important clinical problem in patients with non-transfusion-dependent thalassemia (NTDT), particularly as they advance in age. Current models for iron metabolism in patients with beta (β)-thalassemia intermedia (TI) suggest that suppression of serum hepcidin results in increased iron absorption and release of iron from the reticuloendothelial system, leading to depletion of macrophage iron, relatively low levels of serum ferritin, and liver iron loading. The clinical consequences of iron overload in patients with NTDT are multifactorial and include endocrinopathy, bone disease, thromboembolism, pulmonary hypertension, cerebrovascular and neuronal damage, liver fibrosis or cirrhosis, and increased risk of hepatocellular carcinoma. Although serum ferritin levels correlate with liver iron concentration (LIC), they underestimate iron load in these patients compared with transfusion-dependent patients with equivalent LIC. Therefore, direct measurement of LIC is recommended with chelation therapy as indicated.

A Chinese family carrying novel mutations in SEC23B and HFE2, the genes responsible for congenital dyserythropoietic anaemia II (CDA II) and primary iron overload, respectively

A Chinese family carrying novel mutations in <scp><i>SEC23B</i></scp> and <scp><i>HFE2</i></scp>, the genes responsible for congenital dyserythropoietic anaemia <scp>II</scp> (<scp>CDA II</scp>) and primary iron overload, respectively

Hepcidin is correlated to soluble hemojuvelin but not to increased GDF15 during pregnancy

Increased maternal and foetal iron requirements during pregnancy are compensated by an increase of intestinal iron absorption. Animal studies have shown that the expression of the main iron regulator hepcidin is significantly suppressed during pregnancy, but the factors associated with hepcidin suppression remain unknown. To investigate possible suppressors of hepcidin expression during pregnancy we determined serum concentrations of growth-differentiation factor-15 (GDF15), erythropoietin (EPO), soluble hemojuvelin (HJV) and hepcidin in 42 pregnant women at different time points of gestation and correlated them with serum iron and haematological parameters. Serum iron parameters and serum hepcidin concentration significantly decreased during pregnancy, whereas serum concentrations of GDF15, EPO and soluble HJV significantly increased. A negative correlation of hepcidin with EPO and soluble HJV but no correlation between hepcidin and GDF15 was found. Hepcidin and ferritin were positively correlated throughout the pregnancy. Our findings suggest that hepcidin expression is controlled by body iron stores where soluble HJV and EPO may act as suppressors of hepcidin.

Regulation of hepcidin expression at high altitude

AbstractEnhanced erythropoietic drive and iron deficiency both influence iron homeostasis through the suppression of the iron regulatory hormone hepcidin. Hypoxia also suppresses hepcidin through a mechanism that is unknown. We measured iron indices and plasma hepcidin levels in healthy volunteers during a 7-day sojourn to high altitude (4340 m above sea level), with and without prior intravenous iron loading. Without prior iron loading, a rapid reduction in plasma hepcidin was observed that was almost complete by the second day at altitude. This occurred before any index of iron availability had changed. Prior iron loading delayed the decrease in hepcidin until after the transferrin saturation, but not the ferritin concentration, had normalized. We conclude that hepcidin suppression by the hypoxia of high altitude is not driven by a reduction in iron stores.

Erythropoietin-Driven Signaling Complements the Survival Defect of DMT1-Mutant Erythroid Cells

Abstract 2104Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient’s cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient’s bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient’s results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient’s urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient’s GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells.Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures:No relevant conflicts of interest to declare.

High Levels of Soluble Serum Hemojuvelin in Patients with Congenital Dyserythropoietic Anemia Type I,

Abstract 3179Ineffective erythropoiesis in congenital dyserythropoietic anemia type I (CDA I) is characterized by increased iron absorption mediated by down-regulation of hepcidin. It has been suggested that growth differentiation factor 15 (GDF15) contributes to the inappropriate suppression of hepcidin. Hemojuvelin (HJV), an important regulator of hepcidin production, is highly expressed in skeletal muscle and the liver. Mutations in the gene encoding HJV lead to severe hepcidin deficiency and juvenile hemochromatosis. Membrane-bound HJV (m-HJV) acts as a co-receptor for bone morphogenetic proteins (BMPs). HJV also exists in a soluble form (s-HJV) which competitively down-regulates hepcidin by interfering with BMP signaling. At least two proteases, furin and matriptase-2, cleave m-HJV into s-HJV. Recently, Brasse-Lagnel et al. (Heamtologica 95:2031–7, 2010) reported a new assay for measuring both the furin- and the matriptase-2-cleaved forms of s-HJV. The aim of the present study was to evaluate the possible role of s-HJV in the development of iron overload in CDA I. The study group included eight (8) Israeli Bedouins with CDA I who were homozygous for the founder mutation, c.3124C>T. All were young adults of mean age 27 years. One patient had undergone splenectomy and is currently transfusion-dependent; 3 female patients required occasional blood transfusion during intercurrent infection, pregnancy, and delivery. Nine healthy volunteers served as the comparative group. Laboratory data, including levels of serum hepcidin, ferritin, erythropoietin, soluble transferrin receptor (sTfR), and GDF 15, were previously collected in both groups (Tamary et al. Blood 112:5241–4, 2008). Serum levels of s-HJV were measured in stored frozen samples using a competitive ELISA assay based on an anti-HJV polyclonal antibody (Brasse-Lagnel et al. Heamtologica 95:2031–7, 2010). Compared to the healthy volunteers, the patient group was characterized by significantly higher levels of s-HJV (1.99±0.98mg/L vs 0.36±0.25mg/L; p=0.001), significantly lower level of hemoglobin, and significantly higher levels of serum ferritin, erythropoietin, GDF 15, and sTfR. Serum hepcidin levels were similar in the CDA I patients and the healthy volunteers, but the hepcidin-to-ferritin ratio was significantly lower in the patient group, suggesting that serum hepcidin concentrations were inappropriately low. Levels of s-HJV negatively correlated with hemoglobin levels (R= −0.69 p=0.002) and positively correlated with the iron loading parameter, serum ferritin (R=0.73 p=0.000), and parameters of erythropoiesis, such as serum erythropoietin (R=0.63 p= 0.006) and sTfR (R=0.66 p= 0.008). Additionally, GDF 15 level was positively correlated with s-HJV level (R= 0.64 p=0.006), whereas the hepcidin-to-ferritin ratio (R=−0.797 p=0.000) was negatively correlated with s-HJV level. In summary, our study suggests that s-HJV is overexpressed in patients with CDA I and probably contributes to the down-regulation of hepcidin and the iron-loading pathology. To our knowledge, this is the first study to analyze s-HJV levels in a disease characterized by ineffective erythropoiesis. The main limitation of our study is the small number of patients. Larger studies including patients with CDA and thalassemia are indicated. The source of serum s-HJV and the trigger for its overexpression in CDA I still need to be established. Disclosures:No relevant conflicts of interest to declare.

Pharmacological Suppression of Hepcidin Increases Macrophage Cholesterol Efflux and Reduces Foam Cell Formation and Atherosclerosis

We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.