Duodenal macrophages control dietary iron absorption via local degradation of transferrin.

Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

Iron accumulation in the choroid plexus, ependymal cells and CNS parenchyma in a rat strain with low-grade haemolysis of fragile macrocytic red blood cells.

Iron accumulation in the CNS is associated with many neurological diseases via amplification of inflammation and neurodegeneration. However, experimental studies on iron overload are challenging, since rodents hardly accumulate brain iron in contrast to humans. Here, we studied LEWzizi rats, which present with elevated CNS iron loads, aiming to characterise choroid plexus, ependymal, CSF and CNS parenchymal iron loads in conjunction with altered blood iron parameters and, thus, signifying non-classical entry sites for iron into the CNS. Non-haem iron in formalin-fixed paraffin-embedded tissue was detected via DAB-enhanced Turnbull Blue stainings. CSF iron levels were determined via atomic absorption spectroscopy. Ferroportin and aquaporin-1 expression was visualised using immunohistochemistry. The analysis of red blood cell indices and serum/plasma parameters was based on automated measurements; the fragility of red blood cells was manually determined by the osmotic challenge. Compared with wild-type animals, LEWzizi rats showed strongly increased iron accumulation in choroid plexus epithelial cells as well as in ependymal cells of the ventricle lining. Concurrently, red blood cell macrocytosis, low-grade haemolysis and significant haemoglobin liberation from red blood cells were apparent in the peripheral blood of LEWzizi rats. Interestingly, elevated iron accumulation was also evident in kidney proximal tubules, which share similarities with the blood-CSF barrier. Our data underscore the importance of iron gateways into the CNS other than the classical route across microvessels in the CNS parenchyma. Our findings of pronounced choroid plexus iron overload in conjunction with peripheral iron overload and increased RBC fragility in LEWzizi rats may be seminal for future studies of human diseases, in which similar constellations are found.

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

Simultaneous Genotyping of the rs4762 and rs699 Polymorphisms in Angiotensinogen Gene and Correlation with Iranian CAD Patients with Novel Hexa-primer ARMS-PCR.

BACKGROUND: A significant role of Renin-angiotensin system (RAS) genetic variants in the pathogenesis of essential hypertension and cardiovascular diseases has been proved. This study aimed to develop a new, fast and cheap method for the simultaneous detection of two missense single nucleotide polymorphisms (T207M or rs4762 and M268T orrs699) of angiotensinogen (AGT) in single-step Multiplex Hexa-Primer Amplification Refractory Mutation System - polymerase chain reaction (H-ARMS-PCR). METHODS: In this case-control study, 148 patients with coronary artery disease (CAD) and 135 controls were included. The patients were referred to cardiac centers in Afshar Hospital (Yazd, Iran) from 2012 to 2015. Two sets of inner primer (for each SNP) and one set outer primer pairs were designed for genotyping of rs4762 and rs699 in single tube H-ARMS-PCR. Direct sequencing of all samples was also performed to assess the accuracy of this method. DNA sequencing method validated the results of single tube H-ARMS-PCR. RESULTS: We found full accordance for genotype adscription by sequencing method. The frequency of the AGT T521 and C702 alleles was significantly higher in CAD patients than in the control group (OR: 0.551, 95% CI: 0.359-0.846, P=0.008 and OR: 0.629, 95% CI: 0.422-0.936, P=0.028, respectively). CONCLUSION: This is the first work describing a rapid, low-cost, high-throughput simultaneous detection of rs4762 and rs699 polymorphisms in AGT gene, used in large clinical studies.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.

Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy.

This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.

An open-label trial in Friedreich ataxia suggests clinical benefit with high-dose resveratrol, without effect on frataxin levels.

Friedreich ataxia (FRDA) is due to a triplet repeat expansion in FXN, resulting in deficiency of the mitochondrial protein frataxin. Resveratrol is a naturally occurring polyphenol, identified to increase frataxin expression in cellular and mouse models of FRDA and has anti-oxidant properties. This open-label, non-randomized trial evaluated the effect of two different doses of resveratrol on peripheral blood mononuclear cell (PBMC) frataxin levels over a 12-week period in individuals with FRDA. Secondary outcome measures included PMBC FXN mRNA, oxidative stress markers, and clinical measures of disease severity. Safety and tolerability were studied. Twenty-four participants completed the study; 12 received low-dose resveratrol (1 g daily) and 12 high-dose resveratrol (5 g daily). PBMC frataxin levels did not change in either dosage group [low-dose group change: 0.08 pg/µg protein (95% CI -0.05, 0.21, p = 0.21); high-dose group change: 0.03 pg/µg protein (95% CI -0.10, 0.15, p = 0.62)]. Improvement in neurologic function was evident in the high-dose group [change in Friedreich Ataxia Rating Scale -3.4 points, 95% CI (-6.6, -0.3), p = 0.036], but not the low-dose group. Significant improvements in audiologic and speech measures, and in the oxidative stress marker plasma F2-isoprostane were demonstrated in the high-dose group only. There were no improvements in cardiac measures or patient-reported outcome measures. No serious adverse events were recorded. Gastrointestinal side-effects were a common, dose-related adverse event. This open-label study shows no effect of resveratrol on frataxin levels in FRDA, but suggests that independent positive clinical and biologic effects of high-dose resveratrol may exist. Further assessment of efficacy is warranted in a randomized placebo-controlled trial.

Iron sucrose and ferric carboxymaltose: no correlation between physicochemical stability and biological activity.

Intravenous iron preparations, like iron sucrose (IS) and ferric carboxymaltose (FCM) differ in their physicochemical stability. Thus differences in storage and utilization can be expected and were investigated in a non-clinical study in liver parenchyma HepG2-cells and THP-1 macrophages as models for toxicological and pharmacological target cells. HepG2-cells incorporated significant amounts of IS, elevated the labile iron pool (LIP) and ferritin and stimulated iron release. HepG2-cells had lower basal cellular iron and ferritin content than THP-1 macrophages, which showed only marginal accumulation of IS and FCM. However, FCM increased the LIP up to twofold and significantly elevated ferritin within 24 h in HepG2-cells. IS and FCM were non-toxic for HepG2-cells and THP-1 macrophages were more sensitive to FCM compared to IS at all concentrations tested. In a cell-free environment redox-active iron was higher with IS than FCM. Biostability testing via assessment of direct transfer to serum transferrin did not reflect the chemical stability of the complexes (i.e., FCM > IS). Effect of vitamin C on mobilisation to transferrin was an increase with IS and interestingly a decrease with FCM. In conclusion, FCM has low bioavailability for liver parenchyma cells, therefore liver iron deposition is unlikely. Ascorbic acid reduces transferrin-chelatable iron from ferric carboxymaltose, thus effects on hepcidin expression should be investigated in clinical studies.

Bioavailability and stability of intravenous iron sucrose originator versus generic iron sucrose AZAD.

CONTEXT: Severe iron deficiency requires intravenous iron supplementation to replenish iron stores. Intravenous iron sucrose has been used for decades for the treatment of anemia. New generic iron sucrose products are now marketed for the use in several countries and there is an ongoing discussion about the safety and efficacy of iron sucrose similars. OBJECTIVE: In this study, we compared the iron sucrose originator Venofer® and the generic iron sucrose AZAD (ISA) regarding bioavailability, toxicity and stability in human THP-1 cells and HepG2 cells. METHODS: The bioavailability of Venofer® and ISA was investigated in both cell types by a ferrozin-based assay. The release of incorporated iron was assayed by atomic absorption spectroscopy. Ferritin content was measured by enzyme-linked immunosorbent assay (ELISA). HepG2 cells were used to investigate the intracellular labile iron pool (LIP), which was measured by the fluorescent calcein assay. The amount of redox-active iron within the iron formulations was assayed using fluorescent dichlorofluorescein. RESULTS: We found no significant differences in all parameters between Venofer® and ISA in regard of bioavailability, toxicity and stability in vitro. DISCUSSION: ISA shows identical physico-chemical features and identical bioavailability in vitro. This study is a profound basis for future clinical tests with generic iron sucrose compounds.

Iron deficiency alters megakaryopoiesis and platelet phenotype independent of thrombopoietin.

Iron deficiency is a common cause of reactive thrombocytosis, however, the exact pathways have not been revealed. Here we aimed to study the mechanisms behind iron deficiency-induced thrombocytosis. Within few weeks, iron-depleted diet caused iron deficiency in young Sprague-Dawley rats, as reflected by a drop in hemoglobin, mean corpuscular volume, hepatic iron content and hepcidin mRNA in the liver. Thrombocytosis established in parallel. Moreover, platelets produced in iron deficient animals displayed a higher mean platelet volume and increased aggregation. Bone marrow studies revealed subtle alterations that are suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte ploidy and accelerated megakaryocyte differentiation. Iron deficiency did not alter the production of hematopoietic growth factors such as thrombopoietin, interleukin 6 or interleukin 11. Megakaryocytic cell lines grown in iron-depleted conditions exhibited reduced proliferation but increased ploidy and cell size. Our data suggest that iron deficiency increases megakaryopoietic differentiation and alters platelet phenotype without changes in megakaryocyte growth factors, specifically TPO. Iron deficiency-induced thrombocytosis may have evolved to maintain or increase the coagulation capacity in conditions with chronic bleeding.

White matter changes in patients with friedreich ataxia after treatment with erythropoietin.

BACKGROUND AND PURPOSE: Erythropoietin (EPO) has received growing attention because of its neuroregenerative properties. Preclinical and clinical evidence supports its therapeutic potential in brain conditions like stroke, multiple sclerosis, and schizophrenia. Also, in Friedreich ataxia, clinical improvement after EPO therapy was shown. The aim of this study was to assess possible therapy-associated brain white matter changes in these patients. METHODS: Nine patients with Friedreich ataxia underwent Diffusion Tensor Imaging (DTI) before and after EPO treatment. Tract-based spatial statistics was used for longitudinal comparison. RESULTS: We detected widespread longitudinal increase in fractional anisotropy and axial diffusivity (D||) in cerebral hemispheres bilaterally (P < .05, corrected), while no changes were observed within the cerebellum, medulla oblongata, and pons. CONCLUSIONS: To the best of our knowledge, this is the first DTI study to investigate the effects of EPO in a neurodegenerative disease. Anatomically, the diffusivity changes appear disease unspecific, and their biological underpinnings deserve further study.

Correlation of frataxin content in blood and skeletal muscle endorses frataxin as a biomarker in Friedreich ataxia.

BACKGROUND: Friedreich ataxia is an autosomal recessive disorder caused by mutations in the frataxin gene, leading to reduced levels of the mitochondrial protein frataxin. Assays to quantitatively measure frataxin in peripheral blood have been established. To determine the validity of frataxin as a biomarker for clinical trials, we assessed frataxin in clinically affected tissue. METHODS: In 7 patients with Friedreich ataxia, frataxin content was measured in blood and skeletal muscle before and after treatment with recombinant human erythropoietin, applying the electrochemiluminescence immunoassay. RESULTS: We found frataxin content to be correlated in peripheral blood mononuclear cells and skeletal muscle in drug-naive patients with Friedreich ataxia. The correlation of frataxin content in both compartments remained significant after 8 weeks of treatment. Skeletal-muscle frataxin values correlated with ataxia using the Scale for the Assessment and Rating of Ataxia score. CONCLUSIONS: Our results endorse frataxin measurements in peripheral blood cells as a valid biomarker in Friedreich ataxia.

Effects of erythropoietin on frataxin levels and mitochondrial function in Friedreich ataxia--a dose-response trial.

Friedreich ataxia (FRDA) is an autosomal recessive inherited neurodegenerative disorder leading to reduced expression of the mitochondrial protein frataxin. Previous studies showed frataxin upregulation in FRDA following treatment with recombinant human erythropoietin (rhuEPO). Dose-response interactions between frataxin and rhuEPO have not been studied until to date. We administered escalating rhuEPO single doses (5,000, 10,000 and 30,000 IU) in monthly intervals to five adult FRDA patients. Measurements of frataxin, serum erythropoietin levels, iron metabolism and mitochondrial function were carried out. Clinical outcome was assessed using the "Scale for the assessment and rating of ataxia". We found maximal erythropoietin serum concentrations 24 h after rhuEPO application which is comparable to healthy subjects. Frataxin levels increased significantly over 3 months, while ataxia rating did not reveal clinical improvement. All FRDA patients had considerable ferritin decrease. NADH/NAD ratio, an indicator of mitochondrial function, increased following rhuEPO treatment. In addition to frataxin upregulation in response to continuous low-dose rhuEPO application shown in previous studies, our results indicate for a long-lasting frataxin increase after single high-dose rhuEPO administration. To detect frataxin-derived neuroprotective effects resulting in clinically relevant improvement, well-designed studies with extended time frame are required.

Variations of frataxin protein levels in normal individuals.

Friedreich's ataxia (FRDA) is the most common of the inherited ataxias and is associated with GAA trinucleotide repeat expansions within the first intron of the frataxin (FXN) gene. There are expanded FXN alleles from 66 to 1,700 GAA·TTC repeats in FRDA patients and correlations between number of GAA repeats and frataxin protein levels are assumed. Here, we present for the first time frataxin protein levels as well as analysis of GAA triplet repeats in the FXN gene in a population of 50 healthy Austrian people. Frataxin protein levels were measured in lymphocytes from blood samples by ELISA and GAA repeats were analyzed by capillary electrophoresis. Rather unexpectedly, we found a high variation of frataxin protein levels among the individuals. In addition, there was no correlation between frataxin levels, GAA repeats, age and sex in this group. However, these findings are of great importance for better characterization of the disease.

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae.

BACKGROUND: Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. RESULTS: The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 µm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. CONCLUSIONS: A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

In vitro study on the effects of iron sucrose, ferric gluconate and iron dextran on redox-active iron and oxidative stress.

Concerns exist that administration of intravenous (i.v.) iron preparations is associated with oxidative stress. Therefore iron sucrose (CAS 8047-67-4), ferric gluconate (CAS 34098-81-1) and iron dextran (CAS 9004-66-4) were assessed for redox-active iron by a dichlorofluorescein assay and for intracellular reactive oxygen species (ROS) generation and cytotoxicity in HepG2 cells. Examining each i.v. iron preparation at its maximum concentration achieved following clinically frequently used doses in a 70 kg individual in in vitro experiments, redox-active iron was highest with ferric gluconate, followed by iron dextran and iron sucrose. Interestingly, when the i.v. iron preparations were diluted in human serum instead of buffer, redox-active iron was highest with iron dextran, followed by iron sucrose, and practically disappeared with ferric gluconate. ROS production in HepG2 cells was increased by all i.v. iron preparations. However, in the neutral red cytotoxicity assay all i.v. iron preparations were non-toxic. In conclusion, ferric gluconate showed the highest increase in intracellular ROS-production in HepG2 cells and the highest amount of redox-active iron in buffer in the in vitro assays. In contrast to the other i.v. iron preparations, redox-active iron from ferric gluconate was rendered completely redox-inactive by serum, indicating that redox-active iron in the various preparations has different chemical properties.

Carbamylated erythropoietin increases frataxin independent from the erythropoietin receptor.

BACKGROUND: Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin. Recently we showed in a clinical pilot study in Friedreich's ataxia patients that recombinant human erythropoietin (rhuEPO) significantly increases frataxin-expression. In this in vitro study, we investigated the role of the erythropoietin receptor (EPO-R) in the frataxin increasing effect of rhuEPO and if nonerythropoietic carbamylated erythropoietin (CEPO), which cannot bind to the classical EPO-R increases frataxin expression. MATERIALS AND METHODS: In our experiments human erythroleukaemic K562 cells (+ EPO-R), human monocytic leukemia THP-1 cells (- EPO-R) and isolated primary lymphocytes from healthy control and FRDA patients were incubated with different concentrations of rhuEPO or CEPO. Frataxin-expression was detected by an electrochemical luminescence immunoassay (based on the principle of an ELISA). RESULTS: We show that rhuEPO increases frataxin-expression in K562 cells (expressing EPO-R) as well as in THP-1 cells (without EPO-R expression). These results were confirmed by the finding that CEPO, which cannot bind to the classical EPO-R increased frataxin expression in the same concentration range as rhuEPO. In addition, we show that both EPO derivatives significantly increase frataxin-expression in vitro in control and Friedreich's ataxia patients primary lymphocytes. CONCLUSION: Our results provide a scientific basis for further studies examining the effectiveness of nonerythropoietic derivatives of erythropoietin for the treatment of Friedreich's ataxia patients.

A high throughput electrochemiluminescence assay for the quantification of frataxin protein levels.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients.

Accumulation of mitochondrial genome variations in Persian LQTS patients: a possible risk factor?

BACKGROUND: Long QT syndrome (LQTS) is among arrhythmia disorders of the heart that causes sudden cardiac death in young individuals. As yet, most of investigations have focused on nuclear genome for finding genetic defects in this disorder, but some of the cases with LQTS cannot be explained by mutations of identified genes. On the other hand, it has been reported that the activity of ion channels in cardiomyocytes is sensitive to ATP level. It prompted us to focus on the mitochondrial DNA and monitor the point mutations of genome which are probably the cause of respiratory chain defects and reduced ATP generation. METHODS: We searched about 55% of the mitochondrial DNA (mtDNA) by temporal temperature gradient gel electrophoresis (TTGE), and DNA fragments showing abnormal banding patterns were sequenced for identification of exact mutations. RESULTS: In 39 patients (33 familial and 6 sporadic cases), for the first time, we detected 35 mtDNA mutations in which 8 were novel (23%) and 27 (77%) have been reported in other mitochondrial diseases. Our results showed that these mutations in LQTS patients were higher than those in normal controls (P<.0001), and the number of mutations in LQTS patients with syncope is higher than in patients without syncope (P<.001). CONCLUSIONS: As the mitochondrion's ATP synthesis is important in heart, it is possible that mutations and their accumulation in mtDNA could constitute a predisposing factor that in combination with environmental factors may trigger the syncope in patients with LQTS.