Systemic Iron Overload Research Articles

Hemochromatosis as a cause of central primary Amenorrhea: A literature review and case report

Introduction: Hereditary hemochromatosis (HH) is a genetic disease that causes systemic iron overload. The excess of iron accumulates in several organs, leading to organ dysfunction. Therefore, the excess of iron deposition might create an endocrine impairment, and can further lead to hypogonadism and, rarely, to amenorrhea. In this study, we aim to report a rare cause of HH and primary amenorrhea, with the help of clinical investigations. Case Description: This study is based on a single patient who was diagnosed with amenorrhea. The patient had a previous history of HH, type I diabetes mellitus, and chronic liver disease. The physical and hormonal investigations were normal, and her karyotype was 46 XX. A magnetic resonance imaging (MRI) demonstrated a diffuse reduction of the pituitary tissue signal, which might resemble HH. Discussion: The diagnosis of HH is based on a biochemical and genetic evaluation. The treatment includes regular phlebotomies and the control of comorbidities. In most of the cases, the hypogonadotropic hypogonadism leads to amenorrhea due to a pituitary impairment. However, a rare appearance of amenorrhea without hypogonadism may also be possible. Hence, the investigation of HH should be suggested incases of primary amenorrhea. The early diagnosis of HH is crucial to prevent the morbidity and mortality in patients with these clinical features.

Sideroblastic anemia associated with multisystem mitochondrial disorders.

Sideroblastic anemia represents a heterogeneous group of inherited or acquired diseases with disrupted erythroblast iron utilization, ineffective erythropoiesis, and variable systemic iron overload. In a cohort of 421 patients with multisystem mitochondrial diseases, refractory anemia was found in 8 children. Five children had sideroblastic anemia with increased numbers of ring sideroblasts >15%. Two of the children had a fatal course of MLASA1 syndrome (mitochondrial myopathy, lactic acidosis, and sideroblastic anemia [SA]) due to a homozygous, 6-kb deletion in the PUS1 gene, part of the six-member family of pseudouridine synthases (pseudouridylases). Large homozygous deletions represent a novel cause of presumed PUS1-loss-of-function phenotype. The other three children with SA had Pearson syndrome (PS) due to mtDNA deletions of 4 to 8kb; two of these children showed early onset of PS and died due to repeated sepsis; the other child had later onset of PS and survived as the hematological parameters normalized and the disease transitioned to Kearns-Sayre syndrome. In addition, anemia without ring sideroblasts was found in three other patients with mitochondrial disorders, including two children with later onset of PS and one child with failure to thrive, microcephaly, developmental delay, hypertrophic cardiomyopathy, and renal tubular acidosis due to the heterozygous mutations c.610A>G (p.Asn204Asp) and c.674C>T (p.Pro225Leu) in the COX10 gene encoding the cytochrome c oxidase assembly factor. Sideroblastic anemia was found in fewer than 1.2% of patients with multisystem mitochondrial disease, and it was usually associated with an unfavorable prognosis.

Hepatic hepcidin/intestinal HIF-2α axis maintains iron absorption during iron deficiency and overload.

Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin, a liver-derived hormone that controls iron mobilization through its molecular target ferroportin (FPN), the only known mammalian iron exporter. This pathway is perturbed in diseases that cause iron overload. Additionally, intestinal HIF-2α is essential for the local absorptive response to systemic iron deficiency and iron overload. Our data demonstrate a hetero-tissue crosstalk mechanism, whereby hepatic hepcidin regulated intestinal HIF-2α in iron deficiency, anemia, and iron overload. We show that FPN controlled cell-autonomous iron efflux to stabilize and activate HIF-2α by regulating the activity of iron-dependent intestinal prolyl hydroxylase domain enzymes. Pharmacological blockade of HIF-2α using a clinically relevant and highly specific inhibitor successfully treated iron overload in a mouse model. These findings demonstrate a molecular link between hepatic hepcidin and intestinal HIF-2α that controls physiological iron uptake and drives iron hyperabsorption during iron overload.

Aceruloplasminemia: Waiting for an Efficient Therapy.

Aceruloplasminemia is an ultra-rare hereditary disorder caused by defective production of ceruloplasmin. Its phenotype is characterized by iron-restricted erythropoiesis and tissue iron overload, diabetes, and progressive retinal and neurological degeneration. Ceruloplasmin is a ferroxidase that plays a critical role in iron homeostasis through the oxidation and mobilization of iron from stores and subsequent incorporation of ferric iron into transferrin (Tf), which becomes available for cellular uptake via the Tf receptor. In addition, ceruloplasmin has antioxidant properties preventing the production of deleterious reactive oxygen species via the Fenton reaction. Some recent findings suggest that aceruloplasminemia phenotypes can be more heterogeneous than previously believed, varying within a wide range. Within this large heterogeneity, microcytosis with or without anemia, low serum iron and high serum ferritin, and diabetes are the early hallmarks of the disease, while neurological manifestations appear 10–20 years later. The usual therapeutic approach is based on iron chelators that are efficacious in reducing systemic iron overload. However, they have demonstrated poor efficacy in counteracting the progression of neurologic manifestations, and also often aggravate anemia, thereby requiring drug discontinuation. Open questions remain regarding the mechanisms leading to neurological manifestation and development of diabetes, and iron chelation therapy (ICT) efficacy. Recent studies in animal models of aceruloplasminemia support the possibility of new therapeutic approaches by parenteral ceruloplasmin administration. In this review we describe the state of the art of aceruloplasminemia with particular attention on the pathogenic mechanisms of the disease and therapeutic approaches, both current and perspective.

Correcting Non-Transfusion Dependent β-Thalassemia by Utilizing a Combined Therapy that Modulates EPO Activity by Limiting Erythroid Cellular Iron Intake

Β-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Administration of agents that increase erythropoiesis (such as erythropoietin-Epo) can potentially improve anemia by enhancing the production of red blood cells (RBC). However, this improvement will likely result in exacerbation of splenomegaly and worsening of iron overload due to further suppression of hepcidin, the hormone that controls dietary iron absorption. However, we postulated that, following Epo administration, some level of iron restriction will limit the splenomegaly and iron overload without preventing the improvement of anemia.To increase circulating Epo levels and increase erythropoiesis, we infused animals with primary dermal fibroblasts transduced with a helper dependent adenoviral vector carrying the Epo gene (TARGTEpo). Pharmacokinetic studies indicated that 1e6 cells were sufficient to significantly increase Epo synthesis (up to 47.6%in Hbbth3/+ and 90% in WT). This led to an increase in hemoglobin levels in both mouse models (average of 3-4 g/dL) through the end of the treatment (6 weeks). In Hbbth3/+ mice, Hb levels rose to 10.7±1.0 g/dL in the first week and remained elevated through the end of the 6-weeks of treatment (compared to 7.7±0.7 g/dL in controls). As expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin.To test our hypothesis, we then combined TARGTEpo with low iron diet or antisense oligonucleotides targeting Tmprss6 mRNA (Tmprss6-ASO; the last treatment only for Hbbth3/+ animals). It has already been shown that inhibition of Tmprss6 increases hepcidin expression leading to decreased iron absorption and limited erythroid iron intake, improving anemia, IE, splenomegaly and iron overload in Hbbth3/+ mice (Guo et al, JCI, 2013).In WT animals, after three weeks, the combination of TARGTEpo with low iron diet significantly reduced Hb levels (-40%), RBC number (-38%) and reticulocytes (-80%), when compared to animals overexpressing Epo and receiving a normal iron diet. In contrast, Hbbth3/+ animals on an iron-deficient diet or treated with Tmprss6-ASO in the presence of TARGTEpo showed decreased hemichrome formation and improved anemia through the end of treatment. In particular, Hb levels increased (on average 4 g/dL), reaching levels of 11-12g/dl, corresponding to +36% more than baseline levels in Hbbth3/+ (7.7g/dL) and +25% more compared to Hbbth3/+ mice treated with Tmprss6-ASO alone (9.0g/dL). Animals receiving TARGTEpo with Tmprss6-ASO showed a significant reduction in splenomegaly when compared to baseline levels (-35%) and to animals treated only with TARGTEpo (-52.4%). Preliminary data indicate that organ iron concentration improved or normalized compared to control Hbbth3/+ mice when iron restriction was applied.We then inquired how these two treatments combined improved erythropoiesis. We quantified the number of nucleated erythroid cells positive for the phosphorylated form of pAkt and pJak2, proteins activated by Epo but whose activity is potentially limited by iron restriction. For pAkt+ cells in the spleen, the number of proerythroblasts were increased in Hbbth3/+ treated with TARGTEpo (+35% compared to control Hbbth3/+ mice), while they were reduced in Hbbth3/+ treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO (-72% and -43% respectively, compared to control Hbbth3/+ mice). Other erythroid populations showed a comparable profile for both pAkt+ and pJak2+ nucleated erythroid cells. The total number of progenitor erythroid cells (i.e. BFUe) also followed a similar trend. Cell cycle analysis of splenic erythroid progenitor cells showed a decrease in the percentage of nucleated erythroid cells in mice treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO compared to control or TARGTEpo-treated Hbbth3/+ mice. Given that the number of RBC in circulation was highest in mice treated with TARGTEpo+Tmprss6-ASO, this data suggests that co-administration of these two drugs maximize the ability of each erythroid cell to develop into a functional RBC.In conclusion, we propose that in β-thalassemia, combination of erythropoietic stimulating agents with iron restriction could maximize correction of anemia while improving splenomegaly and iron overload. DisclosuresCasu:Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Neil:Aevi Genomic Medicine, Inc: Employment, Other: Officer and share holder. Guo:Ionis Pharmaceuticals, Inc: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Disc Medicine: Consultancy; Protagonist: Consultancy.

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

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

Iron-enriched diet contributes to early onset of osteoporotic phenotype in a mouse model of hereditary hemochromatosis.

Osteoporosis is associated with chronic iron overload secondary to hereditary hemochromatosis (HH), but the causative mechanisms are incompletely understood. The main objective of this study was to investigate the role of dietary iron on osteoporosis, using as biological model the Hfe-KO mice, which have a systemic iron overload. We showed that these mice show an increased susceptibility for developing a bone loss phenotype compared to WT mice, which can be exacerbated by an iron rich diet. The dietary iron overload caused an increase in inflammation and iron incorporation within the trabecular bone in both WT and Hfe-KO mice. However, the osteoporotic phenotype was only evident in Hfe-KO mice fed the iron-enriched diet. This appeared to result from an imbalance between bone formation and bone resorption driven by iron toxicity associated to Hfe-KO and confirmed by a decrease in bone microarchitecture parameters (identified by micro-CT) and osteoblast number. These findings were supported by the observed downregulation of bone metabolism markers and upregulation of ferritin heavy polypeptide 1 (Fth1) and transferrin receptor-1 (Tfrc), which are associated with iron toxicity and bone loss phenotype. In WT mice the iron rich diet was not enough to promote a bone loss phenotype, essentially due to the concomitant depression of bone resorption observed in those animals. In conclusion the dietary challenge influences the development of osteoporosis in the HH mice model thus suggesting that the iron content in the diet may influence the osteoporotic phenotype in systemic iron overload conditions.

The Functional Versatility of Transferrin Receptor 2 and Its Therapeutic Value.

Iron homeostasis is a tightly regulated process in all living organisms because this metal is essential for cellular metabolism, but could be extremely toxic when present in excess. In mammals, there is a complex pathway devoted to iron regulation, whose key protein is hepcidin (Hepc), which is a powerful iron absorption inhibitor mainly produced by the liver. Transferrin receptor 2 (Tfr2) is one of the hepcidin regulators, and mutations in TFR2 gene are responsible for type 3 hereditary hemochromatosis (HFE3), a genetically heterogeneous disease characterized by systemic iron overload. It has been recently pointed out that Hepc production and iron regulation could be exerted also in tissues other than liver, and that Tfr2 has an extrahepatic role in iron metabolism as well. This review summarizes all the most recent data on Tfr2 extrahepatic role, taking into account the putative distinct roles of the two main Tfr2 isoforms, Tfr2α and Tfr2β. Representing Hepc modulation an effective approach to correct iron balance impairment in common human diseases, and with Tfr2 being one of its regulators, it would be worthwhile to envisage Tfr2 as a therapeutic target.

Air-blood barrier thickening and alterations of alveolar epithelial type 2 cells in mouse lungs with disrupted hepcidin/ferroportin regulatory system.

Iron accumulates in the lungs of patients with common respiratory diseases or transfusion-dependent beta-thalassemia. Based on our previous work, we hypothesized that systemic iron overload affects the alveolar region of the lung and in particular the surfactant producing alveolar epithelial type II (AE2) cells. Mice with a point mutation in the iron exporter ferroportin, a model for human hemochromatosis type 4 were compared to wildtype mice (n = 5 each). Lungs were fixed and prepared for light and electron microscopy (EM) according to state-of-the-art protocols to detect subcellular iron localization by scanning EM/EDX and to perform design-based stereology. Iron was detected as electron dense particles in membrane-bound organelles, likely lysosomes, in AE1 cells. AE2 cells were higher in number but had a lower mean volume in mutated mice. Lamellar body volume per AE2 cell was lower but total volume of lamellar bodies in the lung was comparable to wildtype mice. While the volume of alveoli was lower in mutated mice, the volume of alveolar ducts as well as the surface area, volume and the mean thickness and composition of the septa was similar in both genotypes. The thickness of the air-blood barrier was greater in the mutated than in the WT mice. In conclusion, disruption of systemic iron homeostasis affects the ultrastructure of interalveolar septa which is characterized by membrane-bound iron storage in AE1 cells, thickening of the air-blood barrier and hyperplasia and hypotrophy of AE2 cells despite normal total intracellular surfactant pools. The functional relevance of these findings requires further analysis to better understand the impact of iron on intra-alveolar surfactant function.

Iron chelator deferiprone rescues memory deficits, hippocampal BDNF levels and antioxidant defenses in an experimental model of memory impairment

Brain-derived neurotrophic factor (BDNF) plays a key role in neural development and physiology, as well as in pathological states. Post-mortem studies demonstrate that BDNF is reduced in the brains of patients affected by neurodegenerative diseases. Iron accumulation has also been associated to the pathogenesis of neurodegenerative diseases. In rats, iron overload induces persistent memory deficits, increases oxidative stress and apoptotic markers, and decreases the expression of the synaptic marker, synaptophysin. Deferiprone (DFP) is an oral iron chelator used for the treatment of systemic iron overload disorders, and has recently been tested for Parkinson's disease. Here, we investigated the effects of iron overload on BDNF levels and on mRNA expression of genes encoding TrkB, p75NTR, catalase (CAT) and NQO1. We also aimed at investigating the effects of DFP on iron-induced impairments. Rats received iron or vehicle at postnatal days 12-14 and when adults, received chronic DFP or water (vehicle). Recognition memory was tested 19days after the beginning of chelation therapy. BDNF measurements and expression analyses in the hippocampus were performed 24h after the last day of DFP treatment. DFP restored memory and increased hippocampal BDNF levels, ameliorating iron-induced effects. Iron overload in the neonatal period reduced, while treatment with DFP was able to rescue, the expression of antioxidant enzymes CAT and NQO1.

Non‐transferrin‐bound iron and oxidative stress during allogeneic hemopoietic stem cell transplantation in patients with or without iron overload

Non‐transferrin‐bound iron and oxidative stress during allogeneic hemopoietic stem cell transplantation in patients with or without iron overload

Deletion of hephaestin and ceruloplasmin induces a serious systemic iron deficiency and disrupts iron homeostasis

Multi-copper ferroxidases (MCFs) play important roles in cellular iron metabolism and homeostasis. In this study, we generated the hephaestin (Heph), ceruloplasmin (Cp) single and Heph/Cp double knockout (KO) mice to investigate the roles of MCFs in iron transport among system and vital organs in mice at 4 weeks and 6 months of age. Compared with wild-type (WT) mice, Heph/Cp mice at both ages presented with severe anemia and significantly lower iron level in the serum and spleen, but with significantly higher iron level in the liver, heart, kidney, and duodenal enterocytes. Furthermore, Heph/Cp mice displayed significantly lower level of hepcidin mRNA and transferrin receptor 1 (TFR1) protein expression, but significantly higher level of ferroportin 1 (FPN1) protein expression in the liver than WT mice at 6 months of age. Liver superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities were significantly lower in Heph/Cp KO mice than WT mice at 6 months of age. Together, our results suggest that ablation of HEPH and CP could lead to severe systemic iron deficiency and local tissue iron overload, which disrupt the whole body iron homeostasis and impact on tissue functions.

Calcium channel blockers for preventing cardiomyopathy due to iron overload in people with transfusion-dependent beta thalassaemia.

Beta thalassaemia is a common inherited blood disorder. The need for frequent blood transfusions in this condition poses a difficult problem to healthcare systems. The most common cause of morbidity and mortality is cardiac dysfunction from iron overload. The use of iron chelation therapy has reduced the severity of systemic iron overload but specific, non-toxic treatment is required for removal of iron from the myocardium. To assess the effects of calcium channel blockers combined with standard iron chelation therapy in people with transfusion-dependent beta thalassaemia on the amount of iron deposited in the myocardium, on parameters of heart function, and on the incidence of severe heart failure or arrhythmias and related morbidity and mortality. We searched the Cochrane Haemoglobinopathies Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched ongoing trials databases, and the reference lists of relevant articles and reviews.Date of last search: 24 February 2018. We included randomised controlled trials of calcium channel blockers combined with standard chelation therapy compared with standard chelation therapy alone or combined with placebo in people with transfusion-dependent beta thalassaemia. Two authors independently applied the inclusion criteria for the selection of trials. Two authors assessed the risk of bias of trials and extracted data and a third author verified these assessments. The authors used the GRADE system to assess the quality of the evidence. Two randomised controlled trials (n = 74) were included in the review; there were 35 participants in the amlodipine arms and 39 in the control arms. The mean age of participants was 24.4 years with a standard deviation of 8.5 years. There was comparable participation from both genders. Overall, the risk of bias in included trials was low. The quality of the evidence ranged across outcomes from low to high, but the evidence for most outcomes was judged to be low quality.Cardiac iron assessment, as measured by heart T2*, did not significantly improve in the amlodipine groups compared to the control groups at six or 12 months (low-quality evidence). However, myocardial iron concentration decreased significantly in the amlodipine groups compared to the control groups at both six months, mean difference -0.23 mg/g (95% confidence interval -0.07 to -0.39), and 12 months, mean difference -0.25 mg/g (95% confidence interval -0.44 to -0.05) (low-quality evidence). There were no significant differences between treatment and control groups in serum ferritin (low-quality evidence), liver T2* (low-quality evidence), liver iron content (low-quality evidence) and left ventricular ejection fraction (low-quality evidence). There were no serious adverse events reported in either trial; however, one trial (n = 59) reported mild adverse events, with no statistically significant difference between groups (low-quality evidence). The available evidence does not clearly suggest that the use of calcium channel blockers is associated with a reduction in myocardial iron in people with transfusion-dependent beta thalassaemia, although a potential for this was seen. There is a need for more long-term, multicentre trials to assess the efficacy and safety of calcium channel blockers for myocardial iron overload, especially in younger children. Future trials should be designed to compare commonly used iron chelation drugs with the addition of calcium channel blockers to investigate the potential interplay of these treatments. In addition, the role of baseline myocardial iron content in affecting the response to calcium channel blockers should be investigated. An analysis of the cost-effectiveness of the treatment is also required.

Iron-Overload triggers ADAM-17 mediated inflammation in Severe Alcoholic Hepatitis

Severe alcoholic hepatitis (SAH) is associated with iron accumulation in hepatocytes/macrophages. This possibly correlates with inflammation and stress but the exact mechanism still remains obscure. To understand the role of iron and the mechanisms of systemic iron-overload, a transcriptomic study of liver and Peripheral Blood -Mononuclear-Cells (PBMCs) was undertaken in SAH patients, with and without hepatic iron-overload. Our results show that iron-overload in hepatocytes/macrophages is due to an increased expression of iron-loading receptors and CD163 signaling cascade. Increase in labile iron pool induces expression of iron-loading, oxidative-stress and inflammatory genes along with expression of CD163 and ADAM17. Increased liver iron correlated with circulatory iron, TNF-α, macrophage activation (sCD163) and peroxide-stress in CD163+macrophages in patients who were iron-overloaded and died. Circulatory TNF-α and sCD163 levels were associated with poor outcome. Temporal iron/Fenton stress induced in healthy monocyte-derived-macrophage (MDM)/Tohoku-Hospital-Pediatrics-1(THP1) cells showed higher expression of iron-regulatory, inflammatory and oxidative-stress genes. These genes could be suppressed by iron-chelation. These results suggest that iron mediates inflammation through ADAM17 induction, resulting in macrophage activation and increased shedding of TNF-α and sCD163. These events could be inhibited with iron chelation or with ADAM17-blockade, postulating a therapeutic strategy for SAH patients with iron overload.

Inhibition of Nrf2 alters cell stress induced by chronic iron exposure in human proximal tubular epithelial cells

Iron can catalyze reactive oxygen species (ROS) formation, causing cellular injury. In systemic iron overload, renal tubular epithelial cells are luminally exposed to high iron levels due to glomerular filtration of increased circulating iron. Reports of tubular dysfunction and iron deposition in β-thalassemia major support an association between increased chronic iron exposure and renal tubular injury. In acute iron exposure, Nuclear factor-erythroid 2-related factor 2 (Nrf2) may protect from iron-induced injury, whereas chronic renal stress may lead to Nrf2 exhaustion. We studied the cytotoxic mechanisms of chronic iron exposure using human conditionally immortalized proximal tubular epithelial cells (ciPTECs). Long-term iron exposure resulted in iron accumulation, cytosolic ROS formation and increased heme oxygenase 1 (HMOX-1) mRNA expression (all p < 0.001). This was accompanied by nuclear translocation of Nrf2 and induction of its target protein NQO1, which both could be blocked by the Nrf2 inhibitor trigonelline. Interestingly, iron and trigonelline incubation reduced ROS production, but did not affect HMOX-1 mRNA levels. Moreover, ferritin protein and CHOP mRNA expression were induced in combined iron and trigonelline incubated cells (p < 0.05). Together, these findings suggest that chronic iron exposure induces oxidative stress and that exhaustion of the antioxidant Nrf2 pathway may lead to renal injury.

Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo

Osteoporosis, osteopenia, and pathological bone fractures are frequent complications of iron-overload conditions such as hereditary hemochromatosis, thalassemia, and sickle cell disease. Moreover, animal models of iron overload have revealed increased bone resorption and decreased bone formation. Although systemic iron overload affects multiple organs and tissues, leading to significant changes on bone modeling and remodeling, the cell autonomous effects of excessive iron on bone cells remain unknown. Here, to elucidate the role of cellular iron homeostasis in osteoclasts, we generated two mouse strains in which solute carrier family 40 member 1 (Slc40a1), a gene encoding ferroportin (FPN), the sole iron exporter in mammalian cells, was specifically deleted in myeloid osteoclast precursors or mature cells. The FPN deletion mildly increased iron levels in both precursor and mature osteoclasts, and its loss in precursors, but not in mature cells, increased osteoclastogenesis and decreased bone mass in vivo Of note, these phenotypes were more pronounced in female than in male mice. In vitro studies revealed that the elevated intracellular iron promoted macrophage proliferation and amplified expression of nuclear factor of activated T cells 1 (Nfatc1) and PPARG coactivator 1β (Pgc-1β), two transcription factors critical for osteoclast differentiation. However, the iron excess did not affect osteoclast survival. While increased iron stimulated global mitochondrial metabolism in osteoclast precursors, it had little influence on mitochondrial mass and reactive oxygen species production. These results indicate that FPN-regulated intracellular iron levels are critical for mitochondrial metabolism, osteoclastogenesis, and skeletal homeostasis in mice.

Haemochromatosis: Pathophysiology and the red blood cell1.

Haemochromatosis remains the most prevalent genetic disorder of Caucasian populations in Australia and the United States, occurring in ∼1 of 200 individuals and having a carrier frequency of 10-14%. Hereditary haemochromatosis is an autosomal recessive condition, that is phenotypically characterised by a gradual accumulation of iron, above and beyond that required for biological function. Once the binding capacity of iron carriers reaches saturation, the highly reactive free iron generates radicals that may lead to widespread cellular dysfunction. Thus, the compounding effects of systemic iron overload and the associated oxidative stress in untreated haemochromatosis patients results in tissue damage precipitating severe complications, including: liver cirrhosis, hepatocellular cancer, cardiomyopathy, and diabetes. The primary treatment indicated for individuals with haemochromatosis is venesection therapy (i.e., regular bloodletting of ∼450 mL). Given the frequency of venesection required to decrease and normalise the elevated iron levels, this population may serve as a valuable source of blood products which are in short supply. While the complications associated with elevated iron deposits are frequently reported, the influence of haemochromatosis on the rheological properties of blood and red blood cells (RBC) - major determinants of microvascular blood flow and tissue perfusion - are poorly understood. Limited studies investigating haemorheology in patients with haemochromatosis have reported altered physical properties of blood, which may partly explain the comorbidities associated with the disorder. The current review will explore the aetiology, pathology, and clinical implications of haemochromatosis disease and the associated oxidative stress, with particular emphasis on RBC.

Enhanced labile plasma iron and outcome in acute myeloid leukaemia and myelodysplastic syndrome after allogeneic haemopoietic cell transplantation (ALLIVE): a prospective, multicentre, observational trial

The effect of systemic iron overload on outcomes after allogeneic haemopoietic cell transplantation (HCT) has been a matter of substantial debate. We aimed to investigate the predictive value of both stored (MRI-derived liver iron content) and biologically active iron (enhanced labile plasma iron; eLPI) on post-transplantation outcomes in patients with acute myeloid leukaemia or myelodysplastic syndrome undergoing allogenic HCT. The prospective, multicentre, observational, ALLogeneic Iron inVEstigators (ALLIVE) trial recruited patients at five centres in Germany. We enrolled patients with acute myeloid leukaemia or myelodysplastic syndrome undergoing allogeneic HCT. Patients underwent cytotoxic conditioning for a median of 6 days (IQR 6-7) before undergoing allogeneic HCT and were followed up for up to 1 year (±3 months) post-transplantation. eLPI was measured in serum samples with the FeROS eLPI kit (Aferrix, Tel-Aviv, Israel) and values greater than 0·4 μmol/L were considered to represent raised eLPI. Liver iron content was measured by MRI. The primary endpoints were the quantitative delineation of eLPI dynamics during allogeneic HCT and the correlation coefficient between liver iron content before HCT and dynamic eLPI (eLPIdyn; maximum eLPI minus baseline eLPI). All patients with available data were included in all analyses. This is the final analysis of this completed trial, which is registered with ClinicalTrials.gov, number NCT01746147. Between Dec 13, 2012, and Dec 23, 2014, 112 patients underwent allogeneic HCT. Liver iron content before allogeneic HCT was not significantly correlated with eLPIdyn (ρ=0·116, p=0·22). Serum eLPI concentrations rapidly increased during conditioning, and most (79 [73%] of 108) patients had raised eLPI by the day of transplantation. Patients with a pretransplant liver iron content greater than or equal to 125 μmol/g had an increased incidence of non-relapse mortality (20%, 95% CI 14-26) compared with those with lower concentrations (7%, 2-12; p=0·039) at day 100. Patients who had raised eLPI at baseline also had a significantly increased incidence of non-relapse mortality at day 100 (33%, 15-52) compared with those who had normal eLPI at baseline (7%, 2-13; p=0·00034). eLPI is a possible biological mediator of iron-related toxicity. Peritransplantation eLPI-scavenging strategies could be explored in prospective interventional clinical trials for patients with systemic iron overload. The Technical University of Dresden and Novartis.

Haemochromatosis.

Haemochromatosis is defined as systemic iron overload of genetic origin, caused by a reduction in the concentration of the iron regulatory hormone hepcidin, or a reduction in hepcidin-ferroportin binding. Hepcidin regulates the activity of ferroportin, which is the only identified cellular iron exporter. The most common form of haemochromatosis is due to homozygous mutations (specifically, the C282Y mutation) in HFE, which encodes hereditary haemochromatosis protein. Non-HFE forms of haemochromatosis due to mutations in HAMP, HJV or TFR2 are much rarer. Mutations in SLC40A1 (also known as FPN1; encoding ferroportin) that prevent hepcidin-ferroportin binding also cause haemochromatosis. Cellular iron excess in HFE and non-HFE forms of haemochromatosis is caused by increased concentrations of plasma iron, which can lead to the accumulation of iron in parenchymal cells, particularly hepatocytes, pancreatic cells and cardiomyocytes. Diagnosis is noninvasive and includes clinical examination, assessment of plasma iron parameters, imaging and genetic testing. The mainstay therapy is phlebotomy, although iron chelation can be used in some patients. Hepcidin supplementation might be an innovative future approach.

The Aging of Iron Man.

Brain iron is tightly regulated by a multitude of proteins to ensure homeostasis. Iron dyshomeostasis has become a molecular signature associated with aging which is accompanied by progressive decline in cognitive processes. A common theme in neurodegenerative diseases where age is the major risk factor, iron dyshomeostasis coincides with neuroinflammation, abnormal protein aggregation, neurodegeneration, and neurobehavioral deficits. There is a great need to determine the mechanisms governing perturbations in iron metabolism, in particular to distinguish between physiological and pathological aging to generate fruitful therapeutic targets for neurodegenerative diseases. The aim of the present review is to focus on the age-related alterations in brain iron metabolism from a cellular and molecular biology perspective, alongside genetics, and neuroimaging aspects in man and rodent models, with respect to normal aging and neurodegeneration. In particular, the relationship between iron dyshomeostasis and neuroinflammation will be evaluated, as well as the effects of systemic iron overload on the brain. Based on the evidence discussed here, we suggest a synergistic use of iron-chelators and anti-inflammatories as putative anti-brain aging therapies to counteract pathological aging in neurodegenerative diseases.