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MECCANISMI DI REGOLAZIONE DELL'OMEOSTASI DEL FERRO NEL DIFFERENZIAMENTO ERITROIDE NORMALE E TALASSEMICO
Full text linkINTRODUCTION: Iron homeostasis is maintained in humans trough a meticulous control of intestinal iron absorption, effective utilization of iron by erythropoiesis, efficient recycling of iron from senescente erythrocytes and controlled storage of iron by hepatocytes and macrophages. Little is known about the regulation of iron metabolism in pathological conditions, in particular transfusion independent beta-thalassemia intermedia (TI) transfusion independent and thalassemia major (TM). The clinical manifestations of TI result from three key factors: ineffective erythropoiesis, chronic anemia and iron overload. In patients with thalassemia major (TM), in whom iron loading occurs mainly as a result of transfusion therapy, while in patients with TI accumulate iron primarily due to increased intestinal iron absorption and ineffective erythropoiesis. This leads to an increase in the plasma of iron from tissue and taken up by transferrin, which is saturated with the accumulation of excess free iron. This causes toxicity and tissue damage. The main regulator of iron homeostasis regulation is hepcidin, an hepatic peptide that negatively regulates iron egress from intestinal cells and macrophages by altering the expression of the cellular iron exporter ferroportin. Ferroportin (FPN) is the only mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated. In doing so, hepcidin can control both the total body iron by modulating intestinal iron absorption as well as promote iron available for erythropoiesis by affecting the efficiency of macrophages in recycling iron from effete red blood cells. Despite the key role attributed to hepcidin in the regulation of the iron, the mechanisms that regulate its expression are unknown, in particular it is not known how the increased erythropoietic activity present in TI reduces the expression of hepcidin. A candidate gene involved in this regulation is the GDF15 (growth differentiation factor 15), which is secreted by erythroblasts during erythropoiesis. The only information known about this gene is derived from studies of TM subjects and in vitro studies on cell lines, where it has been observed that in patients with TM, it was observed that the GDF15 is present at high levels in serum and its role may be to inhibit the expression of hepcidin in the liver. It is still unknown why GDF15 is more expressed in beta thalassemia patients than in healthy subjects and how GDF15 can negatively regulates the expression of hepcidin is still unknown.
AIM: To determine the genes expression profile of GDF15, hepcidin and ferroportin isoforms during normal and thalassemic erythroid differentiation in standard cultures and in situations that simulate the iron depletion (deferoxamine) or saturated iron (ferric ammonium citrate), from CD34+ and macrophages of normal and thalassemia intermedia and major subjects.
METHODS: After informed consent, the CD34+ cells and macrophages cells were obtained from peripheral blood of healthy volunteers and from patients with TI and TM by positive and negative respectively selection using anti-CD34-tagged magnetic beads. The CD34+ cells were cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The macrophages cells were cultured for 6 days with a medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) to induce macrophages differentiation. Each culture of CD34+ cells and macrophages was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4M) as iron chelating agent and ferric ammonium citrate (FAC 100 M) at day 0 of culture. The expression profiling of GDF15, hepcidin and ferroportin genes were evaluated at baseline, day 7 and day 14 by real-time PCR (2^-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit.
RESULTS IN CD34+ CELLS: GDF15 expression and secretion increased significantly during erythroid differentiation either in normal, in TI and TM cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were significantly higher compared to control and to TM which had lower values. On the contrary, hepcidin is significantly expressed only in the TM. At day 14 in control cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TI GDF15 expression was down-regulated both by DFO and by FAC. In TM GDF15 expression was down-regulated by DFO and up-regulated by FAC addition. There was the same trend for the secretion of the GDF15 protein. In control cultures, FPN total expression increased significantly during erythroid differentiation, while in TI and TM cultures FPN total was highly expressed at erythroid progenitors stage (day 0 of culture) and decreased at early erythroblasts stage (day 7) and late erythroblasts stage (day 14). In control cultures, FPN1A/FPNTOT was highly expressed at day 0 of culture, decreased significantly at day 7 and increased significantly at day 14. In TM cultures it was expressed at day 0 and decreased both at day 7 and at day 14. In TI cultures, the FPN1A/FPNTOT was highly expressed only day 7. In control cultures the FPN1B/FPNTOT was significantly expressed only at early erythroblasts stage, whereas in TI and TM cultures it was highly expressed at baseline although decreased during differentiation. At day 14 in thalassemia intermedia cultures FPN1B/FPNTOT expression were higher compared to control and to TM. At day 14 in control cultures FPN1A/FPNTOT and FPN1B/FPNTOT expression were not modificated by addition DFO and FAC. In TI and TM cultures, the addition of FAC was not modificated the expression of FPN1A/FPNTOT. In TI it expression was down-regulated by DFO addition, in TM it was up-regulated by DFO. In TM cultures FPN1B/FPNTOT was up-regulated by DFO while it was down-regulated by FAC. In TI FPN1B/FPNOT expression was up-regulated both by DFO and by FAC.
RESULTS IN MACROPHAGES CELLS: In untreated control cultures both FPN1A/FPNTOT and FPN1B/FPNTOT were highly expressed, while they were down-regulated both by DFO and by FAC. In TI and TM cultures FPN1A/FPNTOT and FN1B/FPNTOT were not expressed both in untreated macrophages and in treated macrophages. In control and TI cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TM cultures GDF15 expression was not modificated by addition DFO and FAC.
DISCUSSION: In TI and TM cultures total FPN was highly expressed at erythroid progenitors stage and could contribute to iron overload typical of thalassemia. Conversely, in control cultures the ferroportin was expressed at late erythroblasts stage maybe because the now mature cells does not need iron and made it available to other parts of the body. This correlated with the low levels of hepcidin and with the positive expression of GDF15. In TM cultures the absence of GDF15, the presence of hepcidin and the lack feroportina was due to low concentration of intracellular iron. Instead in TI cultures was much GDF15, a marker of ineffective erythropoiesis, little hepcidin and the good levels of FPN. In TM the ineffective erythropoiesis was suppressed by transfusions. The GDF15 increased during the normal differentiation and thus may play a role in these stages and its expression was modulated by iron levels. In TI and TM the GDF15 was essential for growth but in TI there was no modulation by the iron concentrations. The FPN1A seemed to be important at the beginning and end of erythroid differentiation, while in mid favored retention of iron in erythroblasts for to make hemoglobin. Similar to the situation of the TM but also at the end of erythroid differentiation did not express FPN1A because in reality it had not completed erythropoiesis. In TI the 1A isoform was high at day 7 of culture and low at 14° may be due ineffective erythropoiesis and delayed differentiation. In the control 1B isoform was high at day 7 to escape the repression due to the system IRE / IRP involves the isoform 1A so that, if the body was in conditions of iron deficiency, erythroid cells can export it to ensure the flow to other organs. In TI and TM cells the FPN1B isoform was highly expressed in the early stages of erythroid differentiation, possibly contributing to iron overload in both forms of thalassemia. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression. Control cells treated with an iron chelator or iron did not show changes in the expression of isoform 1A and 1B. The TM cells under conditions of iron depletion increased the expression of FPN1A as the chelator, by subtracting the extracellular iron, created an imbalance of the ion and the cell expressed FPN1A to export it outside. TI cells however, in contact with DFO, decreased the expression of FPN1A because the chelator removed directly to intracellular iron and therefore the cell did not need a transporter. The isoform FPN1B was mostly expressed in TI and TM cells treated with DFO compared to untreated cells, as the decrease caused by the iron chelator, increased the ineffective erythropoiesis and as the 1B represented the ineffective erythropoiesis, it does not could only increase. The untreated control macrophages expressed both isoforms of ferroportin because the recycled iron from macrophages it happened in was two possible ways: it may be stored with the ferritin molecules and used later or exported out of the plasma and therefore needed precisely ferroportin. Conditions of iron depletion or iron saturation, however, strongly down regulated the expression of both isoforms, assuming a more importance of regulation by heme. In TI and TM cells ferroportin was not expressed because the macrophages, affected by extracellular iron overload, repressed the expression of FPN not to be exported more iron potentially toxic. The expression of GDF15 in control macrophages was the same pattern of CD34: conditions of iron depletion increased the expression of GDF15 and conditions of iron saturation decreased its presence. In fact, increased levels of GDF15 caused an increase of iron efflux from macrophages to make it available to others tissues. Decreased levels of GDF15 however, increase the activity of macrophages and helped to retain iron in macrophages for limiting a accumulation of toxic iron. Macrophages TI was the same trend as control macrophages, therefore the regulation of GDF15 was again influenced by the levels of iron. These data suggest that in TI cultures existed two different systems of regulation of GDF15 depending on the type of cell involved: in fact in CD34 cells was an regulation insensitive to variations in iron while in macrophages was an iron-dependent regulation, as in controls cells
Effect of dihydroartemisinin on human Erythroid cell differentiation
No full textWomen in their first pregnancy are at the very high-risk of developing severe malaria which includes maternal anemia, low birth weight of newborns and increased mortality of both mother and infants. The WHO recommends the Intermittent Preventive Treatment to cure malaria during gestation, but drug safety in pregnancy is an issue.
Artemisinin combination therapy is the first line treatment for uncomplicated malaria, but artemisinin derivatives carry a potential toxic effect on embryos. In animal studies they affect embryonic erythroid precursors only on certain days of gestation. This suggests that the target of DHA toxicity could be the primitive erythropoiesis. Our aim was to study the effect of artemisinin and 4-aminoquinoline derivatives on in vitro models which reproduce human erythropoiesis: K562 leukemia cells and CD34+ from human peripheral blood. Cells switch from fetal and embryonic to adult hemoglobin in presence of hemin or butyric acid (K562) or erythropoietin (CD34+). We found that artemisinins inhibit both cell growth and erythroid differentiation (P<0.05), measured as adult hemoglobin synthesis and erythroblasts count. The effect is dose and time-dependent. DHA, which is the active metabolite of artemisinins, has the strongest effect. As expected chloroquine and amodiaquine did not affect cell differentiation, confirming the suitability of the models for studying drug toxicity on developmental erythropoiesis. Moreover, as in animal studies, our results show that a toxic effect of DHA could occur if administered during first trimester of pregnancy, when fetal blood consists mostly of primitive erythroblasts. The support of EU Antimal Project 18834 is acknowledge
Effect of dihydroartemisinin of human erythroid cell differentiation : implications for malaria treatment in pregnancy
No full textBACKGROUND: Severe malaria in pregnancy causes maternal anemia, low birth weight increased mortality of both mother and infants. WHO recommends few antimalarials due to safety problems. Artemisinin combination therapy is the first line treatment, however artemisinin derivatives showed animal embryotoxicity with a reduction of embryonic erythrocytes when treatment is performed on certain days of gestation. AIMS: To investigate the effect of Dihydroartemisinin (DHA), the metabolite of artemisinins, on an in vitro model reproducing human erythropoiesis and to characterize the erythroid target stage, in order to predict the window of susceptibility to DHA in human pregnancy. METHODS: The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. DHA at 0,5 or 2 ÂμM, according to the dosages of previous animal experiments, was added for the first time at day 0 (on isolated stem cell), at day 2 (on early erythroid progenitors), at day 4 (in presence of both early progenitors and pro-erythroblasts), at day 7 (on basophilic erythroblasts) or at day 11 (polychromatic erythroblasts) then continuously every 3 days up to 14 days, because of its short half life. Cells growth and viability were evaluated by trypan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glycophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific gene expression analysis with real-time PCR. RESULTS: DHA was added on stem cells or early erythroid progenitors caused a transient inhibition of both cell growth and differentiation up to day 7, but then the treated cells started growing and completed their erythroid differentiation at day 14 of culture. When DHA was added on basophilic erythroblasts, a significant and long lasting effect decrease in proliferation as well as a delay in erythroid differentiation was observed. Up to day 14. DHA added on mature stages i.e. polychromatic erythroblasts, only a small reduction of cell growth has been observed without any consequence for the erythroid cell differentiation. CONCLUSIONS: These data suggest that DHAâ s specific target is the basophilic erythroblast, since DHA added at this stage causes a significant inhibition of erythroid differentiation. Based on these in vitro results, we hypothesize that DHA could affect human primitive erythropoiesis, which occurs during the late phase of human secondary yolk sac erythropoiesis (weeks 4-8 of gestation), when foetal blood is formed of only primitive erythroblasts. This means that if the treatment with DHA or artemisinin derivatives is performed during the first trimester of human pregnancy, toxic effects on embryo could be expected
In vitro ferroportin expression in thalassemia intermedia during erythroid differentiation
No full textINTRODUCTION: Ferroportin (FPN) is the sole mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated.
AIM: To investigate the different expression profile of ferroportin isoforms during erythroid differentiation in control and TI cell cultures.
METHODS: After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with thalassemia intermedia by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The expression profiling of FPN1A and FPN1B was evaluated at baseline, day 7 and day 14 of culture by real-time PCR (-dCt).
RESULTS: In control cultures, FPN1A isoform was highly expressed at erythroid progenitors stage (day 0 of culture), decreased at early erythroblasts stage (day 7) and increased again at late erythroblasts stage (day 14). In TI cultures, the FPN1A isoform expression remained high even in early erythroblasts (Table 1). In control cultures the FPN1B isoform expression was very low at any stage of erythroid differentiation, whereas in TI cultures it was highly expressed at baseline and, althoug decreased during differentiation, remained always higher than control (Table 2).
CONCLUSIONS: In thalassemic conditions the FPN1B is the major expressed ferroportin isoform, possibly contributing to iron overload. In control cultures, FPN1A was mainly expressed in undifferentiated erythroid progenitors and in mature erythroblasts, suggesting a functional role at these stages of erythroid differentiation. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expressio
DHA inhibits human erythroid cell differentiation by altering the GATA switch
No full textWHO recommends to avoid artemisinin treatment during the first trimester of pregnancy, because animal models showed a significant depletion of embryonic red cells, which occurs only during a specific days of gestation. We recently demonstrated for the first time that DHA, which is the
in vivo metabolite of many artemisinin derivatives, inhibits human erythroid cell differentiation, as well. We showed that DHA specifically targets the pro and basophilic erythroblasts during
in vitro erythroid cell differentiation of CD34+ stem cells. By using K562 cells differentiated toward the erythroid lineages by chemical inducers and by comparing the effects of several
artemisinins, we confirmed that DHA is the most toxic compound of this drug family. Significant reduction by DHA was observed not only of cell growth, but also of erythroid cell maturation, as shown by the changes in cell viability, cell cycle progression, GpA expression, inhibition of γ-globin
gene and GATA-1 mRNAs. In addition, we observed that the toxicity is related to pathways which regulate the haemoglobin synthesis. In fact, DHA rapidly induces the release of Cytochrome C from the mitochondria, which in turn, activates Caspase-3 and, together with the HSP70 down
regulation, induces the GATA-1 cleavage and the up-regulation of GATA-2. In conclusion, altering the GATA switch, DHA modifies the fate of the erythroid cell: it prevents the erythroid cell differentiation and simultaneously causes the arrest of cell growth and, eventually, the cell death through apoptosis. This dual effect is clearly dose-dependent. In conclusion, our results support WHO recommendations and the urgent need to better define the risk-benefit of the use of artemisinins treatment for malaria during the first trimester of human pregnancy
Growth differentiation factor 15 expression and regulation during erythroid differentiation in non-transfusion dependent thalassemia syndromes
No full text
Seven novel genetic mutations within the 5 ' UTR and the housekeeping promoter of HMBS gene responsible for the non-erythroid form of acute intermittent porphyria (vol 49, pg 147, 2012)
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Seven novel genetic mutations within the 5′UTR and the housekeeping promoter of HMBS gene responsible for the non-erythroid form of acute intermittent porphyria
No full textAcute intermittent porphyria (AIP) is an autosomal dominant disorder caused by molecular abnormalities in the HMBS gene. This gene is transcribed from two promoters to produce ubiquitous and erythroid specific isoforms of porphobilinogen deaminase (PBGD). In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Only one mutation sited in the housekeeping promoter has been previously reported as causative for this form of AIP. In this study, we identified one small deletion and six nucleotide substitutions within the 5'UTR and the housekeeping promoter of HMBS gene: c.1-440_-427del14bp; c.1-421G>A; c.1-331C>T; c.1-270G>A; c.1-122T>A; c.1-103C>T; c.1-28A>C. Using luciferase reporter assays and quantitative PCR experiments, we characterized the functional role of these seven novel genetic variants demonstrating that all mutations cause a significant loss of transcriptional activity. Our investigations suggest that these nucleotide substitutions may alter critical binding sites for transcriptional factors, which confirms that these regions represent an important molecular target for pathogenesis of non-erythroid form of acute intermittent porphyri
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