Iron-overloaded Mice Research Articles

Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice.

Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation.

Decrease of Peripheral Monocyte Relative Number and Mean Platelet Volume in Iron Overloaded Mice

Iron overload (IO) because of multiple blood transfusion as a definite therapy for hematological disease with chronic and severe anemia has become a major concern. Deleterious complication contributed by chemically reactive deregulated iron may affect cellular homeostasis systemically lead to tissue and organ damage. When this toxicity occurred in blood cells, alteration of peripheral hematological profile concerning erythrocyte, leucocyte, and platelet most likely to be modified and imperatively need to be evidenced. The experimental IO mice model was established by injecting a low and high dose of iron dextran intraperitoneally. Peripheral erythrocyte, leucocyte and platelet indices measured by hematology analyzer were analyzed. A dynamic tendency of leucocyte absolute cell number and differential cell count of low and high dose iron treatment and a significant decrease of differential monocyte count were found. In addition, high dose iron treatment showed a significantly lower mean platelet volume. In conclusion, this study verified that IO impaired the cellular hematological indices by selectively suppress monocyte number addressing that this mononuclear phagocyte was the most affected immune cell. Furthermore, low mean platelet volume following acquired platelet function defect was evidenced. This research provided an animal experimental model that could be used for further study in finding alternative therapeutic targets on the pathophysiology of iron overload diseases, such as thalassemia.

Iron is Responsible for Production of Reactive Oxygen Species Regulating Vasopressin Expression in the Mouse Paraventricular Nucleus.

Reactive oxygen species (ROS) act as signaling molecules for maintaining homeostasis, particularly in the regulation of body-fluid balance in the paraventricular nucleus (PVN) of the hypothalamus. However, there has been little discussion regarding the source of ROS generation in this hypothalamic region. Because iron is the most abundant metal in the brain, we hypothesized that iron may act as a source of ROS, which regulate vasopressin (VP) expression. In the present study, we compared the amount of iron in the PVN to that in other forebrain regions of normal ICR mice, and examined the relationship among iron, ROS, and VP in the PVN of the iron-overloaded with iron dextran and iron-chelated mice with deferoxamine. The amount of iron in the PVN was significantly higher than in any of the forebrain regions we examined. The amount of iron in the PVN was significantly increased in iron-overloaded mice, although not in iron-chelated mice. These results suggest that the PVN exhibits high iron affinity. Both ROS production and VP expression in the PVN of iron-overloaded mice were significantly increased relative to levels observed in control mice. VP concentration in blood of iron-overloaded mice was also significantly higher than that of control mice. Interestingly, iron overload did not alter the expression of nitric oxide synthase, another modulator of VP expression. Taken together, our results suggest that high levels of iron in the PVN induce the production of ROS, which regulate VP expression, independent of nitric oxide signaling.

μXRF and LA-ICP-TQMS for quantitative bioimaging of iron in organ samples of a hemochromatosis model

Hereditary hemochromatosis is the most common autosomal recessive genetic disorder of the iron metabolism. Iron accumulation in various organs, especially in liver and pancreas leads to diseases and may cause organ failure. In this study, methods for elemental bioimaging by means of quantitative micro X-ray fluorescence analysis (μXRF) and laser ablation-inductively coupled plasma-triple quadrupole mass spectrometry (LA-ICP-TQMS) were developed and applied to investigate the pathophysiological development of iron accumulation in murine tissue based on animals with an iron-overload phenotype caused by a hepatocyte-specific genetic mutation. The use of an external calibration with matrix-matched gelatin standards enables the quantification of iron by means of μXRF without the typically used fundamental parameters method or Monte Carlo simulation, which becomes more imprecise when analyzing thin tissue sections. A fast, non-destructive screening of the iron concentration and distribution with a spatial resolution of 25 μm in liver samples of iron-overload mice was developed. For improved limits of detection and higher spatial resolution down to 4 μm, LA-ICP-TQMS was used with oxygen as reaction gas. By monitoring the mass shift of 56Fe to 56Fe16O, a limit of detection of 0.5 μg/g was obtained. With this method, liver and pancreas samples of iron-overload mice as well as control mice were successfully analyzed. The high spatial resolution enabled the analysis of the iron distribution in different liver lobules. Compared to the established Prussian blue staining, both developed methods proved to be superior due to the possibility of direct iron quantification in the tissues.

Alpha-lipoic acid attenuates iron-overload-induced structural changes in the liver of the laboratory mouse (Mus musculus)

Objectives: The role of alpha-lipoic acid in the amelioration of iron overload-induced hepatic damage remains largely under-explored. Therefore, this paper aimed at describing the structural effects of alpha lipoic acid on the liver following iron overload in mice. Methods: After ethical approval, a total of 24 male mice were used. Twenty mice were randomly divided into 2 groups: A and B. Group A rats received 50mg/kg of iron dextran intraperitoneally daily for 49 days while those in group B received a daily oral dose of 100mg/kg alpha lipoic acid by gavage in addition to the treatment in A. Four mice were used as normal controls. At the endpoint of the experiment, the livers were harvested and studied for iron deposition, parenchymal histoarchitecture and hepatocyte densities. Photomicrographs were taken using a digital photomicroscope for morphometric analysis. Results: Treatment of mice with iron led to a distortion of the histoarchitecture of the liver which was attenuated with co-administration of alpha lipoic acid. Additionally, co-treatment of iron with alpha lipoic acid resulted in significant lowering of hepatic iron deposition (p<0.001), reduction in leukocyte infiltration and significantly greater hepatocyte densities (p<0.001). Conclusion: Alpha-lipoic acid appreciably attenuates the structural damage in the liver induced by iron overload.

Green tea extract modulates oxidative tissue injury in beta-thalassemic mice by chelation of redox iron and inhibition of lipid peroxidation

Iron overload in patients with β-thalassemia can cause oxidative organ dysfunction. Iron chelation along with antioxidant supplementation can ameliorate such complications and prolong lives. Green tea extract (GTE) rich in epigallocatechin-3-gallate (EGCG) exhibits anti-oxidation and iron chelation properties in β-knockout thalassemic (BKO) mice diagnosed with iron overload. We investigated the effects of GTE and deferiprone (DFP) alone in combination with one another, and upon the levels of redox-active iron, lipid-peroxidation product, insulin and hepcidin in BKO mice. A state of iron overload was induced in the mice via a trimethylhexanoyl-ferrocene supplemented (Fe) diet for 3 months, and the mice were treated daily with either: DFP (50 mg/kg), DFP (50 mg/kg) plus GTE (50 mg EGCG equivalent/kg), or GTE alone for 2 months. Plasma non-transferrin bound iron (NTBI), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepcidin and insulin; tissue iron and MDA were measured. DFP, GTE and GTE + DFP effectively decreased plasma MDA (p < 0.05), NTBI and ALT, and increased plasma hepcidin and insulin. All the treatments also reduced iron accumulation and MDA production in both the pancreas and liver in the mice. However, the combination therapy demonstrated no advantages over monotherapy. The findings suggest GTE improved liver and pancreatic β-cell functions in iron-overloaded β-thalassemia mice by diminishing redox iron and free radicals, while inhibiting lipid peroxidation. Consequently, there are indications that GTE holds significant potential for clinical use.

ALK3 undergoes ligand-independent homodimerization and BMP-induced heterodimerization with ALK2

The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are essential for expression of hepcidin, a key iron regulatory hormone. In mice, hepatocyte-specific Alk2 deficiency leads to moderate iron overload with periportal liver iron accumulation, while hepatocyte-specific Alk3 deficiency leads to severe iron overload with centrilobular liver iron accumulation and a more marked reduction of basal hepcidin levels. The objective of this study was to investigate whether the two receptors have additive roles in hepcidin regulation. Iron overload in mice with hepatocyte-specific Alk2 and Alk3 (Alk2/3) deficiency was characterized and compared to hepatocyte-specific Alk3 deficient mice. Co-immunoprecipitation studies were performed to detect the formation of ALK2 and ALK3 homodimer and heterodimer complexes in vitro in the presence and absence of ligands. The iron overload phenotype of hepatocyte-specific Alk2/3-deficient mice was more severe than that of hepatocyte-specific Alk3-deficient mice. In vitro co-immunoprecipitation studies in Huh7 cells showed that ALK3 can homodimerize in absence of BMP2 or BMP6. In contrast, ALK2 did not homodimerize in either the presence or absence of BMP ligands. However, ALK2 did form heterodimers with ALK3 in the presence of BMP2 or BMP6. ALK3-ALK3 and ALK2-ALK3 receptor complexes induced hepcidin expression in Huh7 cells. Our data indicate that: (I) ALK2 and ALK3 have additive functions in vivo, as Alk2/3 deficiency leads to a greater degree of iron overload than Alk3 deficiency; (II) ALK3, but not ALK2, undergoes ligand-independent homodimerization; (III) the formation of ALK2-ALK3 heterodimers is ligand-dependent and (IV) both receptor complexes functionally induce hepcidin expression in vitro.

Iron Overload Exacerbates the Risk of Hemorrhagic Transformation After tPA (Tissue-Type Plasminogen Activator) Administration in Thromboembolic Stroke Mice.

Background and Purpose- Recanalization with tPA (tissue-type plasminogen activator) is the only pharmacological therapy available for patients with ischemic stroke. However, the percentage of patients who may receive this therapy is limited by the risk of hemorrhagic transformation (HT)-the main complication of ischemic stroke. Our aim is to establish whether iron overload affects HT risk, to identify mechanisms that could help to select patients and to prevent this devastating complication. Methods- Mice fed with control or high-iron diet were subjected to thromboembolic stroke, with or without tPA therapy at different times after occlusion. Blood samples were collected for determination of malondialdehyde, matrix metalloproteinases, and fibronectin. Brain samples were collected 24 hours after occlusion to determine brain infarct and edema size, hemorrhage extension, IgG extravasation, and inflammatory and oxidative markers (neutrophil infiltration, 4-hydroxynonenal, and matrix metalloproteinase-9 staining). Results- Despite an increased rate of recanalization, iron-overload mice showed less neuroprotection after tPA administration. Importantly, iron overload exacerbated the risk of HT after early tPA administration, accelerated ischemia-induced serum matrix metalloproteinase-9 increase, and enhanced basal serum lipid peroxidation. High iron increased brain lipid peroxidation at most times and neutrophil infiltration at the latest time studied. Conclusions- Our data showing that iron overload increases the death of the compromised tissues, accelerates the time of tPA-induced reperfusion, and exacerbates the risk of HT may have relevant clinical implications for a safer thrombolysis. Patients with stroke with iron overload might be at high risk of HT after fibrinolysis, and, therefore, clinical studies must be performed to confirm our results.

Iron excess upregulates SPNS2 mRNA levels but reduces sphingosine-1-phosphate export in human osteoblastic MG-63 cells.

Systemic iron excess favors the development of osteoporosis, especially during genetic hemochromatosis. The cellular mechanisms involved are still unclear despite numerous data supporting a direct effect of iron on bone biology. Therefore, the aim of this study was to characterize mechanisms involved in the iron-related osteoblast impairment. We studied, by using the MG-63 cell lines, the effect of iron excess on SPNS2 gene expression which was previously identified by us as potentially iron-regulated. Cell-type specificity was investigated with hepatoma HepG2 and enterocyte-like Caco-2 cell lines as well as in iron-overloaded mouse liver. The SPNS2-associated function was also investigated in MG-63 cells by fluxomic strategy which led us to determinate the S1P efflux in iron excess condition. We showed in MG-63 cells that iron exposure strongly increased the mRNA level of the SPNS2 gene. This was not observed in HepG2, in Caco-2 cells, and in mouse livers. Fluxomic study performed concomitantly on MG-63 cells revealed an unexpected decrease in the cellular capacity to export S1P. Iron excess did not modulate SPHK1, SPHK2, SGPL1, or SGPP1 gene expression, but decreased COL1A1 and S1PR1 mRNA levels, suggesting a functional implication of low extracellular S1P concentration on the S1P/S1PR signalizing axis. Our results indicate that iron impacts the S1P/S1PR signalizing axis in the MG-63 cell line and suggest that iron can affect the bone-associated S1P pathway and favor the occurrence of osteoporosis during chronic iron overload.

Renally‐Clearable Polymeric Nanochelator for Iron Overload Therapy

Iron (Fe) is an essential metal, but high Fe stores are toxic and cause heart and liver failures, arthritis, and diabetes. Since there is no recognized active pathway for Fe excretion, disposing extra Fe from the body is the primary therapeutic goal of treating patients with iron overload (IO). While the chelation therapy has been widely used to alleviate IO, Fe chelators have serious adverse effects, such as hypotension, gastrointestinal bleeding, kidney failure and liver fibrosis, likely due to non‐specific distribution in off‐target tissues. Consequently, there is an unmet need for a new therapeutic strategy for IO. Here we developed an ultrasmall nanoparticle (NP) that covalently binds to an FDA‐approved Fe chelator deferoxamine (DFO), which limits the distribution into off‐target tissues, while efficiently capturing excess Fe. We first generated renally‐clearable ɛ‐poly‐L‐lysine (EPL) conjugated with zwitterionic near‐infrared fluorophore ZW800. We then produced DFO‐coated NP by conjugating four DFO molecules on the surface of each EPL with balanced charges (DFO4‐NP). The ferrozine competition assay demonstrated that the association constant of DFO4‐NP to Fe was greater by a 3.5‐fold (p&lt;0.001) compared to native DFO, indicating improved Fe binding affinity after conjugation. Next, in vivo chelation efficacy of DFO4‐NP (2 μmol as NP/kg) was determined in IO mice induced by intraperitoneal injection of Fe dextran (100 mg/kg). Urinary Fe content collected over 4 h post‐dose was increased by a 2.5‐fold (p&lt;0.001) and by an 8.6‐fold (p&lt;0.001) after intravenous (i.v.) and subcutaneous (s.c.) injection of DFO4‐NP, respectively, compared to blank NP. Similarly, renal clearance of Fe was increased by a 2.8‐fold (p&lt;0.001) and a 9.5‐fold (p&lt;0.001) after i.v. and s.c. of DFO4‐NP, respectively, compared to NP alone. Moreover, when compared to equimolar dose of native DFO, DFO4‐NP showed increased urinary recovery and renal clearance of Fe by a 2.8‐fold (p=0.003) and by a 2.5‐fold (p=0.003), respectively. These suggest the greater chelation efficiency and urinary excretion of Fe by s.c. DFO‐NP administration than by native DFO or blank NP. We further evaluated the efficacy of DFO4‐NP after s.c. doses daily for 5 d in dietary IO mice. DFO4‐NP decreased non‐heme Fe content in serum (22%, p=0.010), liver (26%, p=0.011) and spleen (26%, p=0.011) compared to blank NP. Consistently, DFO4‐NP decreased the status of an Fe‐storage protein ferritin (Ftn), a reliable marker of body Fe stores, in both liver (28%, p=0.041) and spleen (35%, p=0.024), compared to blank NP. These results indicate that DFO‐NPs efficiently remove excess Fe stores from plasma and tissues after repeated administrations. Moreover, H&amp;E staining showed a decreased density of tubular cells in the kidney after native DFO treatment, while no kidney damage was observed in mice treated with NP alone or DFO4‐NP. Taken together, the renally‐clearable DFO‐NPs provide improved therapeutic efficacy against physiological complications resulting from IO disorders, while minimizing the inherent toxicity of small molecule chelators.Support or Funding InformationNIH U54 HL119145, American Heart Association17GRNT33460134This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MyD88 Adaptor Protein Is Required for Appropriate Hepcidin Induction in Response to Dietary Iron Overload in Mice.

Iron homeostasis is tightly regulated to provide virtually all cells in the body, particularly red blood cells, with this essential element while defending against its toxicity. The peptide hormone hepcidin is central to the control of the amount of iron absorbed from the diet and iron recycling from macrophages. Previously, we have shown that hepcidin induction in macrophages following Toll-like receptor (TLR) stimulation depends on the presence of myeloid differentiation primary response gene 88 (MyD88). In this study, we analyzed the regulation of iron metabolism in MyD88−/− mice to further investigate MyD88 involvement in iron sensing and hepcidin induction. We show that mice lacking MyD88 accumulate significantly more iron in their livers than wild-type counterparts in response to dietary iron loading as they are unable to appropriately control hepcidin levels. The defect was associated with inappropriately low levels of Smad4 protein and Smad1/5/8 phosphorylation in liver samples found in the MyD88−/− mice compared to wild-type mice. In conclusion, our results reveal a previously unknown link between MyD88 and iron homeostasis, and provide new insights into the regulation of hepcidin through the iron-sensing pathway.

Effects of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac function and Ca2+ regulation in iron-overloaded thalassemic mice

Although disturbance of cardiac Ca2+ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca2+ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca2+ transients and Ca2+ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca2+ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca2+-ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca2+ transients including decreased intracellular Ca2+ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca2+ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation.

Combined iron chelator and T-type calcium channel blocker exerts greater efficacy on cardioprotection than monotherapy in iron-overload thalassemic mice

Although both iron chelators and T-type calcium channel (TTCC) blockers have been shown to exert cardioprotection by decreasing cardiac iron deposition and reducing left ventricular (LV) dysfunction via different channels in iron-overloaded rodent models, the cardioprotective effects of combined iron chelator and TTCC blocker treatment in thalassemic mice has not been investigated. We hypothesized that a combined iron chelator and TTCC blocker exerts better cardioprotection than monotherapy by decreasing cardiac iron accumulation, apoptosis and oxidative stress. An iron-overload condition was induced in heterozygous βKO thalassemic (HT) mice and wild-type (WT) mice by high iron diet consumption (FE) for 3 months. Then, the iron chelator deferiprone (DFP), the TTCC blocker efonidipine (Efo), and combined DFP plus Efo were fed via oral gavage for 1 month whilst the high iron diet was continued. LV function, heart rate variability (HRV), apoptosis and cardiac iron accumulation were determined. Chronic iron-overload in mice led to increased cardiac iron deposition, oxidative stress, apoptosis, and impaired LV function and HRV. Although DFP and Efo showed similar cardioprotective efficacy, the combined DFP plus Efo therapy exerted greater efficacy in reducing cardiac iron deposition and cellular apoptosis than either of the monotherapies in iron-overload thalassemic mice.

Nanogel-DFO conjugates as a model to investigate pharmacokinetics, biodistribution, and iron chelation in vivo

Deferoxamine (DFO) to treat iron overload (IO) has been limited by toxicity issues and short circulation times and it would be desirable to prolong circulation to improve non-transferrin bound iron (NTBI) chelation. In addition, DFO is currently unable to efficiently target the large pool of iron in the liver and spleen. Nanogel-Deferoxamine conjugates (NG-DFO) can prove useful as a model to investigate the pharmacokinetic (PK) properties and biodistribution (BD) behavior of iron-chelating macromolecules and their overall effect on serum ferritin levels. NG-DFO reduced the cytotoxicity of DFO and significantly reduced cellular ferritin levels in IO macrophages in vitro. PK/BD studies in normal rats revealed that NG-DFO displayed prolonged circulation and preferential accumulation into the liver and spleen. IO mice treated with NG1-DFO presented significantly lower levels of serum ferritin compared to DFO. Total renal and fecal elimination data point to the need to balance prolonged circulation with controlled degradation to accelerate clearance of iron-chelating macromolecules.

Advanced iron-overload cardiomyopathy in a genetic murine model is rescued by resveratrol therapy.

Iron-overload cardiomyopathy is prevalent on a worldwide basis and is a major comorbidity in patients with genetic hemochromatosis and secondary iron overload. Therapies are limited in part due to lack of a valid preclinical model, which recapitulates advanced iron-overload cardiomyopathy. Male hemojuvelin (HJV) knockout (HJVKO) mice, which lack HJV, a bone morphogenetic co-receptor protein required for hepcidin expression and systemic iron homeostasis, were fed a high-iron diet starting at 4 weeks of age for a duration of 1 year. Aged HJVKO mice in response to iron overload showed increased myocardial iron deposition and mortality coupled with oxidative stress and myocardial fibrosis culminating in advanced iron-overload cardiomyopathy. In a parallel group, iron-overloaded HJVKO mice received resveratrol (240 mg/day) at 9 months of age until 1 year of age. Echocardiography and invasive pressure–volume (PV) loop analyses revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. In addition, myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCa2a) levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCa2a levels and suppressed up-regulation of the sodium–calcium exchanger (NCX1). Further, iron-mediated oxidative stress and myocardial fibrosis were suppressed by resveratrol treatment with concomitant activation of the p-Akt and p-AMP-activated protein kinase (AMPK) signaling pathways. A combination of ageing and high-iron diet in male HJVKO mice results in a valid preclinical model that recapitulates iron-overload cardiomyopathy in humans. Resveratrol therapy resulted in normalization of cardiac function demonstrating that resveratrol represents a feasible therapeutic intervention to reduce the burden of iron-overload cardiomyopathy.

Green synthesis of safe zero valent iron nanoparticles by Myrtus communis leaf extract as an effective agent for reducing excessive iron in iron-overloaded mice, a thalassemia model.

Green synthesis of Myrtus communis-Zero Valent Iron Nanoparticles (MC-ZVINs) was carried out in an alkaline environment. These nanoparticles were introduced for testing in vivo iron chelation in an iron-overload thalassemia model in mice. The MC-ZVINs were characterized by the use of imaging (electron microscopy) and spectroscopic (UV-vis and infrared absorption, dynamic light scattering and X-ray diffraction) methods. These methods demonstrated that some polyphenols are bound to the surfaces of the MC-ZVINs as a capping/stabilizing agent. The MC-ZVINs and defroxamine were injected 5 times a week over 4 weeks. The total iron and Fe3+ content of serum was determined by microwave plasma-atomic emission spectroscopy and an Fe3+ ion measuring kit respectively and liver enzyme levels were evaluated with an auto-analyzer. Morphological changes of the liver were examined by haematoxylin and eosin and Prussian blue staining. Additionally, the bioactivity of the iron nanoparticles was checked by DPPH and nitric oxide radical scavenging activity, reducing power and iron (Fe2+) chelating ability. Iron-overloaded mice treated with the MC-ZVINs, indicated a drastic reduction in serum iron content when compared with the control groups and those treated with defroxamine. MC-ZVINs exhibit satisfactory potency to chelate excessive iron in mice. The data collected in this study will be helpful for selecting the most appropriate operational conditions for synthesizing green ZVINs in biomedical applications.

A combination of an iron chelator with an antioxidant exerts greater efficacy on cardioprotection than monotherapy in iron-overload thalassemic mice

Many recent studies have shown that antioxidant compounds decrease cardiac oxidative stress, decrease cardiac iron deposition, and improve cardiac dysfunction in iron-overload induced cardiomyopathy in animal models. Interestingly, a therapy including the combination of the iron chelator deferiprone (DFP) plus the antioxidant N-acetylcysteine (NAC) has been shown to significantly decrease oxidative stress and restore heart and brain function in iron-overloaded rats. However, the cardioprotective effects of this combined DFP and NAC treatment in thalassemic mice have not been investigated. We hypothesised that the combination of DFP and NAC exerts better cardioprotection than monotherapy via decreasing cardiac iron accumulation, oxidative stress, and apoptosis in thalassemic mice. The iron-overload condition was induced in heterozygous βKO HT and wild-type mice by instigating high iron diet consumption (FE) for three months. Then, iron chelator DFP (75 mg/kg/day twice a day), antioxidant NAC (100 mg/kg/day once a day), and combined DFP plus NAC were fed via oral gavage for one month with continuous iron feeding. Left ventricular (LV) function, heart rate variability (HRV), apoptosis, and cardiac iron accumulation were determined. Chronic iron-overload in mice led to increased cardiac iron deposition, oxidative stress, apoptosis, and impaired LV function and HRV. Although DFP and NAC showed similar cardioprotective efficacy, combined DFP plus NAC exerted greater efficacy in reducing both cardiac iron deposition and cellular apoptosis than monotherapy. In conclusion, combined iron chelator and NAC treatment exert the greatest cardioprotective efficacy when compared with either of the monotherapies in iron-overload thalassemic mice.

HDAC1 Governs Iron Homeostasis Independent of Histone Deacetylation in Iron-Overload Murine Models.

Iron-overload disorders are common and could lead to significant morbidity and mortality worldwide. Due to limited treatment options, there is a great need to develop novel strategies to remove the excess body iron. To discover potential epigenetic modulator in hepcidin upregulation and subsequently decreasing iron burden, we performed an epigenetic screen. The in vivo effects of the identified compounds were further tested in iron-overload mouse models, including Hfe-/-, Hjv-/-, and hepatocyte-specific Smad4 knockout (Smad4fl/fl;Alb-Cre+) mice. Entinostat (MS-275), the clinical used histone deacetylase 1 (HDAC1) inhibitor, was identified the most potent hepcidin agonist. Consistently, Hdac1-deficient mice also presented higher hepcidin levels than wild-type controls. Notably, the long-term treatment with entinostat in Hfe-/- mice significantly alleviated iron overload through upregulating hepcidin transcription. In contrast, entinostat showed no effect on hepcidin expression and iron levels in Smad4fl/fl;Alb-Cre+ mice. Further mechanistic studies revealed that HDAC1 suppressed expression of hepcidin through interacting with SMAD4 rather than deacetylation of SMAD4 or histone-H3 on the hepcidin promoter. The findings uncovered HDAC1 as a novel hepcidin suppressor through complexing with SMAD4 but not deacetylation of either histone 3 or SMAD4. In addition, our study suggested a novel implication of entinostat in treating iron-overload disorders. Based on our results, we conclude that entinostat strongly activated hepcidin in vivo and in vitro. HDAC1 could serve as a novel hepcidin suppressor by binding to SMAD4, effect of which is independent of BMP/SMAD1/5/8 signaling. Antioxid. Redox Signal. 28, 1224-1237.

Heme oxygenase-1 protects bone marrow mesenchymal stem cells from iron overload through decreasing reactive oxygen species and promoting IL-10 generation

Iron overload (IO) caused by frequent blood transfusion in hematological diseases has become a major concern. In this study, up-regulation of heme oxygenase-1 (HO-1), a protector against oxidative stress, was observed in bone marrow mesenchymal stem cells (BMMSCs) at the early stage of IO and had favorable prognosis in an IO mouse model. Given that the protective role of HO-1 in IO damage of BMMSCs was still unknown, the mechanism was explored in vitro and in vivo. BMMSCs were transfected with HO-1/siHO-1 in vitro, and the mouse model was established to further evaluate the effect of HO-1 on IO in vivo. As a result, HO-1 decreased the apoptotic rate of BMMSCs with IO through reducing intracellular reactive oxygen species (ROS) but increasing IL-10 secretion. In addition, IL-10 was mediated by HO-1 via the ERK pathway. Intracellular iron was down-regulated by hepcidin depending on IL-10. In conclusion, HO-1 protects BMMSCs from ROS by secreting IL-10 upon iron overload.

Immunoassay for human serum erythroferrone

AbstractErythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin (EPO). We previously demonstrated that ERFE messenger RNA expression and serum protein concentration increase in mice subjected to hemorrhage or EPO therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in mice with nontransfused β-thalassemia. We now report the development and technical validation of a rabbit monoclonal antibody–based sandwich immunoassay for human ERFE. We use this assay to show that blood loss or EPO administration increases serum ERFE concentrations in humans, and that patients with both nontransfused and transfused β-thalassemia have very high serum ERFE levels, which decrease after blood transfusion. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis.