scholarly journals Iron Chelation Improves Erythropoiesis in a Mouse Model of Myelodysplastic Syndrome

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2335-2335
Author(s):  
Wenbin An ◽  
Maria Feola ◽  
Marc Ruiz Martinez ◽  
Amit Verma ◽  
Yelena Ginzburg

Abstract Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis, cytopenias, and transformation to acute leukemia (AML). Low risk MDS patients exhibit a longer median survival, the lowest rate of progression to AML, and account for approximately two-thirds of all MDS patients, requiring recurrent RBC transfusions to alleviate symptomatic anemia. Transfusion-dependence results in iron overload which is associated with reduced overall survival. Methods to diagnose and treat (e.g. iron chelator deferiprone (DFP)) iron overload are available, but because retrospective studies to date only identify a correlative, not a causal, relationship between iron overload and reduced survival, there is little consensus on whether benefits of its diagnosis and treatment outweigh risks in this patient population. We aimed to evaluate and characterize the effect of iron loading and iron chelation on ineffective hematopoiesis in NUP98-HOXD13 transgenic (NHD13) mice, a highly penetrant model with peripheral blood cytopenias and ineffective hematopoiesis with dysplasia, consistent with MDS in humans [Lin Blood 2005]. We hypothesize that iron overload in MDS has deleterious biological effects on hematopoiesis with increased likelihood of worsening disease, reversible with iron chelation therapy. Our preliminary data demonstrates that 1) DFP enhances erythroid differentiation in CD34+ cells from MDS patients; and 2) exogenous iron suppresses erythropoiesis in wild type (WT) mice. The current experiments reveal the effect of exogenous iron and DFP in NHD13 mice. Age and gender matched 5 month old mice, 5-10 mice per group, were injected with 20mg iron dextran and/or treated with DFP (175 mg/mouse) in the drinking water over 4 weeks. Treated mice were analyzed and compared with PBS injected NHD13 mice and WT controls. Our experiments demonstrate that NHD13 mice exhibit anemia and leukopenia, increased serum erythropoietin (Epo) concentration, and higher MCV despite no difference in reticulocyte count relative to WT controls (Table I) and no splenomegaly (Fig 1a). In addition, erythroblast ROS concentration (Fig 1b) and iron load in the liver (Fig 1c) increase without differences in transferrin saturation or hepcidin expression in NHD13 relative to WT mice. These findings demonstrate characteristics consistent with human MDS patients. We then analyzed the effects of additional iron loading, iron chelation, or a combination of both on parameters of iron metabolism and erythropoiesis in NHD13 mice. Iron-treated NHD13 mice exhibit higher WBC count, RBC count, and hemoglobin, both DFP- and iron-treated NHD13 mice exhibit decreased serum Epo, and DFP+iron-treated NHD13 mice exhibit decreased serum Epo while increasing RBC count and hemoglobin (Table I). These findings suggest that exogenous iron increases Epo responsiveness and extramedullary erythroid mass in NHD13 mice in a manner similar to what we observed in iron-treated thalassemic mice [Ginzburg Exp Hem 2009]. Furthermore, spleen size increased (Fig 1a) and erythroblast ROS decreased (Fig 1b) only in iron-treated NHD13 mice. These findings suggest that erythroblast ROS is unrelated to excess iron in NHD13 mice. Liver iron increased in iron-treated and decreased in DFP-treated NHD13 mice (Fig 1c) as expected. In addition, CD71 (TfR1) surface expression on bone marrow erythroblasts is suppressed in all treated NHD13 mice but only significantly decreased in DFP-treated mice (Fig 1d), suggesting expected iron restriction. Lastly, DFP- and DFP+iron-treated (but not iron-treated) NHD13 mice decreased the proportion of bone marrow erythroblasts (Fig 1e) and increased erythroid differentiation in NHD13 mouse bone marrow (Fig 1f). Taken together, our findings demonstrate the robustness of NHD13 mice as a model of MDS to study erythropoiesis, the utility of iron injection in NHD13 mice to mimic robust iron overload in MDS, and the effectiveness of DFP in enhancing erythroid differentiation, reversal of erythroid expansion, and Epo responsiveness in NHD13 mice. Additional experiments (i.e. RBC survival, analysis of bone marrow signaling, erythroferrone expression, and parameters of the iron restriction response) to further explore the dysregulation of iron metabolism in NHD13 mice are ongoing. Disclosures No relevant conflicts of interest to declare.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-24-SCI-24
Author(s):  
Yelena Ginzburg

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


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 122-122 ◽  
Author(s):  
Lap Shu Alan Chan ◽  
Lilly Chunhong Gu ◽  
Michael J. Rauh ◽  
Richard A. Wells

Abstract Abstract 122 Introduction: Transfusion-related iron overload is common in MDS. Iron catalyzes, via the Fenton reaction, excess production of reactive oxygen species (ROS), which are known to cause cell senescence and death, promote DNA damage and accelerate carcinogenesis. In addition to the well characterized effects of iron overload on the heart, liver, and endocrine organs, clinical data have suggested iron also causes haematopoietic toxicity in MDS, since iron chelation can lead to dramatic reduction in transfusion requirements and in large registry studies leukaemia-free survival is inversely related to serum ferritin. These observations have led to controversy since they are supported by no mechanistic or animal model data. Hypothesis: We have demonstrated that iron overload increases intracellular ROS (iROS) in early haematopoietic cells in MDS. We hypothesize that iron, via increased iROS, promotes accumulation of DNA damage in MDS HSCs and thus, in the context of the genomic instability of the MDS clone, accelerates progression of MDS to AML. Here we report the results of experiments that establish the biological and mechanistic plausibility of this hypothesis. Results: To establish that the B6D2F1 mouse model, which has been used in studies of cardiac and hepatic iron overload, is also a suitable model of bone marrow iron overload, mice (n=5 per cohort) were given iron dextran (0-150 mg i.p.) and sacrificed 3 days later. Severe weight loss was noted in the iron-loaded animals. Iron deposition was confirmed by Prussian Blue staining in the bone marrow, liver, myocardium, and the red pulp of the spleen. The cardiac effects of this degree of iron overload compromise survival, preventing assessment of longer-term effects of iron on haematopoiesis. We therefore evaluated the effects of lower doses of iron dextran (0, 5, 10, or 20 mg; n=5 per cohort) over 3 months. Increased iROS was seen in lineage negative (lin−) CD45+ bone marrow cells for animals that received 5 mg iron. However, iROS levels decreased progressively from the 10–20 mg treated animals, possibly representing an increase in apoptosis in early haematopoietic cells exposed to the greatest oxidative stress. Consistent with this, we observed increased apoptosis in early erythroid progenitors for the 20 mg iron treated animals (p<0.05). We adapted the chronic iron overload mouse model to evaluate the effect of iron overload on leukaemogenesis. B6D2F1 mice were sublethally irradiated (300 cGy) followed by s.c. injection of 0.5 mg dexamethasone, a protocol which induces a pre-leukaemic state leading, in SJL mice, to AML in 50–75% with a 12 month latency. These mice were then loaded with 0 or 5 mg I.P. iron dextran, n=6 per cohort). Three mice from each cohort were sacrificed and analyzed 3 months after iron loading. Expansion of the splenic white pulp was observed in iron loaded mice and flow cytometric analysis of the bone marrow cells revealed expansion of the lin− CD45+ early haematopoietic population. Furthermore, in one iron loaded mouse we observed a lin− CD45lo population with size and complexity similar to that of haematopoietic progenitors, suggesting blast accumulation. The remaining mice (n=3 per cohort) continued to be observed. One mouse in the iron loaded cohort died eight months after iron loading. Post-mortem examination revealed severe hepatomegaly and splenomegaly, massive splenic and hepatic infiltration by leukaemic blasts, and extensive bone marrow necrosis, fibrosis, and substantial blast accumulation. To establish a plausible mechanism for the promotion of leukaemia development by iron, we tested the ability of iron to cause DNA damage in a haematopoietic cell line. HL60 cells line were treated with ferric ammonium sulfate (10 or 100 μ M) and DNA damage was assessed by flow cytometry for γH2AX, an indicator of DNA double-strand breaks. Elevated γH2AX was observed in HL60 cells 2 hours after iron loading, and sustained DNA damage was noted till the end of the experiment at day 4. Conclusions: Our observations demonstrate that iron is mutagenic in haematopoietic cells and can promote progression of a pre-leukaemic state to frank AML. We postulate that iron is not itself leukaemogenic, but, by causing DNA damage, promotes clonal evolution in MDS. Further evaluation in animal models and in clinical trials is necessary to elucidate the clinical implications of these observations, especially in regard to the deployment of iron chelation therapy. Disclosures: No relevant conflicts of interest to declare.


2019 ◽  
Vol 41 (28) ◽  
pp. 2681-2695 ◽  
Author(s):  
Francesca Vinchi ◽  
Graca Porto ◽  
Andreas Simmelbauer ◽  
Sandro Altamura ◽  
Sara T Passos ◽  
...  

Abstract Aims Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE−/− FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). Methods and results Compared with normo-ferremic ApoE−/− mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE−/− FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE−/− FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. Conclusion Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-37-SCI-37
Author(s):  
Elizabeta Nemeth ◽  
Tomas Ganz ◽  
Léon Kautz

For successful expansion of erythropoiesis, the activity of the hormone erythropoietin (EPO) must be coordinated with the supply of iron to erythroid precursors. Increased iron supply for erythropoiesis is ensured by the suppression of hepcidin, the iron-regulatory hormone produced by the liver. Low hepcidin levels allow greater absorption of dietary iron and greater mobilization of iron from the stores in the spleen and the liver. The mechanisms coordinating erythropoietic activity with iron delivery are not well understood. We recently identified erythroferrone as a new mediator of hepcidin suppression during stress erythropoiesis1. Erythroferrone (ERFE) is a member of the C1q/TNF-related protein (CTRP) family of metabolic mediators. ERFE is produced in response to EPO by erythroblasts of the bone marrow and spleen of mice. The induction of ERFE by EPO was dependent on Jak2/Stat5 signaling. Ex vivo treatment of human erythroblasts with EPO also resulted in a dramatic induction of ERFE expression. The essential role of ERFE in acute hepcidin suppression by erythropoiesis was demonstrated in ERFE-deficient mice. In contrast to wild-type mice which suppressed hepcidin ~10-fold within hours after hemorrhage or erythropoietin injection, no hepcidin suppression was observed in ERFE knockout mice within 24 h. As a consequence, ERFE-deficient mice exhibited delayed recovery of hemoglobin after hemorrhage or severe inflammation. Treatment of mice or hepatocytes with recombinant ERFE protein confirmed the hepcidin-suppressive activity of the protein. It remains to be seen whether administration of ERFE protein would be useful for the treatment of anemia of inflammation mediated by elevated hepcidin. In iron-loading anemias including β-thalassemia, hepcidin is chronically suppressed by the exuberant but ineffective erythropoietic activity. This is the cause of iron overload in untransfused thalassemia patients and may contribute to iron loading even in transfused patients. We found that ERFE expression is greatly increased in the bone marrow and spleen of mice with β-thalassemia intermedia (th3 model). Transgenic ablation of ERFE in th3 mice normalized hepcidin and partially corrected their iron overload. Although human studies of the role of ERFE in health and disease are clearly needed, ERFE is a promising candidate for the pathological suppressor of hepcidin in anemias with ineffective erythropoiesis. References: 1. Kautz L, Jung G, Valore EV, et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014; 46: 678-684. Disclosures Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Merganser Biotech: Equity Ownership. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Keryx Pharma: Consultancy; Merganser Biotech: Consultancy, Equity Ownership.


2020 ◽  
Author(s):  
shujuan zhou ◽  
Lan Sun ◽  
Shanhu Qian ◽  
Yongyong Ma ◽  
Ruye Ma ◽  
...  

Abstract Background: Iron overload is common in patients with haematological disorders, and it is known to have a suppressive effect on haematogenesis. However, the mechanism by which iron overload affects haematogenesis is still unclear. The antioxidant curcumin has been reported to protect against iron overload-induced bone marrow damage, although the mechanism underlying this protective effect remains to be elucidated.Methods: We established iron overload cell and mouse models. Mitochondrion-derived reactive oxygen species (mROS) levels, autophagy levels, and the SIRT3/SOD2 pathway were examined in these models and in the bone marrow of patients with iron overload.Results: Iron overload was shown to depress haematogenesis and induce mitochondrion-derived superoxide anion-dependent autophagic cell death. Iron loading decreased SIRT3 protein expression, promoted an increase in SOD2, and led to the elevation of mROS. These effects were reversed by the overexpression of SIRT3. Curcumin treatment ameliorated peripheral blood cells, enhanced SIRT3 activity, decreased SOD2 acetylation, inhibited mROS production, and suppressed iron loading-induced autophagy.Conclusions: These results suggest that curcumin exerts a protective effect on bone marrow by reducing mROS-stimulated autophagic cell death in a manner dependent on the SIRT3/SOD2 pathway.


2015 ◽  
Vol 22 (2) ◽  
pp. 128 ◽  
Author(s):  
D. Sanford ◽  
C.C. Hsia

Patients with myelodysplastic syndrome (mds) experience clinical complications related to progressive marrow failure and have an increased risk of developing acute myeloid leukemia. Frequent red blood cell transfusion can lead to clinical iron overload and is associated with decreased survival in mds patients. Iron chelation therapy reduces markers of iron overload and prevents end-organ damage.Here, we present the case of a patient with lowrisk mds with transfusional iron overload. He was treated for 2 years with an oral iron chelator, deferasirox, and after 12 months of treatment, he experienced a hemoglobin increase of more than 50 g/L, becoming transfusion-independent. He has remained transfusion-independent, with a normal hemoglobin level, for more than 2 years since stopping chelation therapy. Hematologic and erythroid responses have previously been reported in mds patients treated with iron chelation. The durability of our patient’sresponse suggests that iron chelation might alter the natural history of mds in some patients.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2685-2685 ◽  
Author(s):  
Lap Shu Alan Chan ◽  
Rena Buckstein ◽  
Marciano D. Reis ◽  
Alden Chesney ◽  
Adam Lam ◽  
...  

Abstract Introduction: The biology of myelodysplastic syndrome (MDS) is poorly understood, and treatment options are limited. Thus, most MDS patients require chronic red blood cell transfusion, and many develop secondary iron overload. Although the pathophysiological consequences of iron overload to the heart, liver, and endocrine organs have been well characterized, its effects on haematopoiesis have not been studied. However, it has been observed that chelation therapy in iron-overloaded MDS patients may result in reduction of transfusion requirements, and recent studies have suggested a correlation between the use of iron chelation therapy and improvement in leukaemia-free survival in MDS. At the cellular level, iron toxicity is mediated in large part via the generation of reactive oxygen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haematopoietic stem cells, and that ROS cause DNA damage and promote the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature. Hypothesis: We hypothesize that iron overload secondary to transfusion leads to increased levels of intracellular ROS in early haematopoeitic cells in MDS. The increase in intracellular ROS in MDS would be predicted to lead further impairment of haematopoiesis via stem cell exhaustion and while promoting accumulation of DNA damage by myelodysplastic stem cells and early progenitors, thus accelerating progression of MDS to acute leukaemia. Results: To test this hypothesis, we examined the relationship between transfusion-related iron overload and ROS content of CD34+ bone marrow cells in MDS. ROS content was measured in CD34+ cells by flow cytometry in bone marrow aspirates from 34 consecutive MDS patients (CMML=4, MDS/MPD=2, RA=4, RARS=3, RCMD=2, RAEB 1=6, RAEB 2=12, RAEB-t/AML=1). The patients represented a wide range of prior transfusion burden (0-&gt;300 units PRBC) and serum ferritin levels (11-&gt;10000 μg/L). ROS was strongly correlated with serum ferritin concentration for patients with iron overload (serum ferritin &gt;1000 μg/L; n=14, R=0.733, p&lt;0.005). The correlation between ROS and ferritin level was even stronger in the subset of patients with RAEB 1 or RAEB 2 and iron overload (n=11, R=0.838, p&lt;0.005). In contrast, no correlation between ROS and ferritin level was demonstrated for patients with serum ferritin &lt;1000 μg/L (n=20). Importantly, iron chelation therapy was associated with a reduction in CD34+ cell ROS content in one patient. To assess the effect of iron overload on normal stem cell and progenitor function, we established a mouse model of subacute bone marrow iron overload. B6D2F1 mice were loaded with iron dextran by intraperitoneal injection (150mg total iron load over 21 days), and sacrificed three days after the end of iron loading. Iron staining of tissue sections confirmed iron deposition in the bone marrow, liver, and myocardium. The development of splenomegaly was noted in iron-loaded animals. Flow cytometric analysis revealed increased apoptosis of bone marrow cells in iron loaded mice based on annexin V+/7 AAD-staining (6.26±0.96% versus 3.54±0.99% for control mice, paired student’s t-Test p&lt;0.005). However, ROS content in CD117+ progenitors of iron loaded mice was similar to control mice. Thus, subacute iron loading in mice increases apoptosis but does not alter the ROS content of HSCs; we postulate that chronic iron overload is required to achieve this effect. Conclusions: These results establish a relationship between CD34+ cell ROS content and serum ferritin concentration in MDS patients with iron overload, and indicate that iron chelation therapy in this patient population reverses this ROS accumulation. The physiological consequences of this relationship are currently being investigated in this patient set by haematopoietic colony assays and assessment of DNA damage in CD34+ cells. Nonethelesss, these data may have key implications for the deployment of iron chelation therapy in MDS patients, and may explain the association between the use of iron chelation and improved leukaemia-free survival in MDS.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2059-2059
Author(s):  
Maya Otto-Duessel ◽  
Casey Brewer ◽  
Aleya Hyderi ◽  
Jens Lykkesfeldt ◽  
Ignacio Gonzalez-Gomez ◽  
...  

Abstract Abstract 2059 Introduction: Iron dextran injections are often used to induce iron overload in rodents, for the purposes of assessing iron chelation therapy. In gerbils, we have previously described that deferasirox therapy preferentially clears hepatocellular iron when compared with reticuloendothelial stores. Ascorbate deficiency, which is common in humans with iron overload, produces similar profound disparities between reticuloendothelial and parenchymal iron stores. We postulated that iron-induced ascorbate deficiency might be exaggerating reticuloendothelial iron retention in gerbils receiving deferasirox therapy. This study examined the effect of supplemental ascorbate on spontaneous iron loss and deferasirox chelation efficiency in the iron-dextran loaded gerbil. Methods: 48 female gerbils underwent iron dextran loading at 200 mg/kg/week for 10 weeks. Sixteen animals were sacrificed at 11 weeks to characterize iron loading; eight were on standard rodent chow and eight had chow supplemented with 2250 ppm of ascorbate. 32 additional animals that were not ascorbate supplemented during iron loading transitioned into the chelation phase. Half were subsequently placed on ascorbate supplemented chow and both groups were assigned to receive either deferasirox 100 mg/kg/day five days per week or sham chelation. Animals received iron chelation for twelve weeks. Liver histology was assessed using H & E and Prussian blue stains. Iron loading was ranked and graded on a five-point scale by an experienced pathologist screened to the treatment arm. Iron quantitation in liver and heart was performed by atomic absorption. Results: Table 1 one summarizes the findings. During iron dextran loading, ascorbate supplementation lowered wet weight liver iron concentration but not liver iron content suggesting primarily changes in tissue water content. Spontaneous iron losses were insignificant, regardless of ascorbate therapy. Deferasirox lowered liver iron content 56% (4.7% per week) in animals without ascorbate supplementation and 48.3% (4.0% per week) with ascorbate supplementation (p=NS). Cardiac iron loading, unloading and redistribution were completely unaffected by ascorbate supplementation. Spontaneous iron redistribution was large (1.9% – 2.3% per week). Deferasirox chelation did not lower cardiac iron to a greater degree than spontaneous cardiac iron redistribution. Histologic grading paralleled tissue wet weight iron concentrations. Ascorbate treatment lowered the rank and absolute iron score in liver during iron loading (p=0.003) and there was a trend toward lower iron scoring in sham treated animals (p=0.13). Ascorbate had no effect on histological score or relative compartment distributions of iron in deferasirox chelated animals (p=0.5). Ascorbate supplementation was sufficient to increase total plasma ascorbate levels from 25 ± 12.2 uM to 38.4 ± 11 uM at 10 weeks (p=0.03). In the liver, ascorbate increased from 1203 ± 212 nmol/g of tissue to 1515 ± 194 nmol/g of tissue (p=0.01) with supplementation. No significant change in total ascorbate was observed in the heart. Discussion: We hypothesized that ascorbate supplementation might improve reticuloendothelial iron accessibility to deferasirox by facilitating redox cycling. Although gerbils synthesize their own ascorbate, supplementation was able to raise both serum and hepatic total ascorbate levels. However, increasing ascorbate did not change either the amount or distribution of tissue iron in deferasirox-treated animals. Disclosures: Nick: Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2423-2423
Author(s):  
Jean Hendy ◽  
Stewart A Fabb ◽  
Meaghan Wall ◽  
Lorraine J Gudas ◽  
Grant A. McArthur ◽  
...  

Abstract Abstract 2423 Homeobox (Hox) genes have been shown to play critical roles in normal and aberrant hematopoiesis, however, little is known about the role of Hoxa1. Unlike most Hox genes, Hoxa1 is expressed as two different isoforms: full length Hoxa1 (FL-Hoxa1) and a truncated form, Hoxa1-T, resulting from alternative splicing within exon 1 of the gene. The two isoforms are expressed differentially in immature hematopoietic cells. Overexpression of FL-Hoxa1 and Hoxa1-T in mouse bone marrow (BM) cells significantly increased, and significantly decreased, respectively, cell proliferation in vitro compared to control (MXIE) transduced BM cells. These findings suggested that Hoxa1-T may negatively regulate FL-Hoxa1. We therefore generated a mutant Hoxa1 (muHoxa1) that expresses FL-Hoxa1 but was no longer capable of generating Hoxa1-T by changing the AGC serine to a TCT serine at the splice site of Hoxa1-T. Hoxa1 levels in muHoxa1-overexpressing BM were significantly higher than those in FL-Hoxa1 BM. Furthermore, BM cells overexpressing muHoxa1 had higher proliferative potential in vitro than FL-Hoxa1-overexpressing BM. Their in vivo potential was therefore assessed. At 12 weeks post-transplant, all primary transplant recipients had similar %GFP expression in the peripheral blood (PB), being approximately 10%. At this time point all recipients of muHoxa1-overexpressing BM cells displayed thrombocytopenia (mean ± SEM PB platelets (x 106/ml): MXIE= 1062 ± 75; FL-Hoxa1= 1053 ± 146; muHoxa1= 681 ± 71*, *P<0.05 vs MXIE). Secondary transplants were performed into irradiated and non-irradiated recipients. Strikingly, the %GFP+ve cells were markedly increased in the PB of recipients of muHoxa1 BM (%GFP: MXIE: 0.29 ± 0.06; FL-Hoxa1: 3.33 ± 1.38; muHoxa1: 27.46 ± 8.35*; *P<0.05 vs MXIE and FL-Hoxa1). All secondary recipients of muHoxa1 BM developed myeloid neoplasias, resembling myelodysplastic syndrome (MDS). The thrombocytopenia persisted (PB platelets (x 106/ml): MXIE: 923 ± 42; FL-Hoxa1: 812 ± 38; muHoxa1: 388 ± 108*; *P<0.001 vs MXIE and FL-Hoxa1) and secondary recipients of muHoxa1 BM developed anemia (PB Hb (g/L): MXIE: 143 ± 2.6; FL-Hoxa1: 146 ± 2.6; muHoxa1: 117 ± 5.2*, *P<.0005 vs MXIE and FL-Hoxa1). The PB leukocyte counts in the majority of muHoxa1 recipients were unchanged compared to MXIE and FL-Hoxa1 recipients, however, muHoxa1 PB cells were predominantly granulocytes. These neoplasms also occurred in non-irradiated recipients of muHoxa1 BM, although they had a much longer latency. Interestingly, 40% of the non-irradiated recipients developed acute myeloid leukemia between 6 and 12 months post-transplant. Importantly, integration site and cytogenetic analysis demonstrated that the malignant phenotype was not due to co-operating insertional mutagenesis or chromosomal instability in the transduced cells. The PB anemia was accompanied by a significant two-fold reduction in Ter119+ cells in BM of muHoxa1-overexpressing cells (GFP+ve= 16.3± 5.3%, GFP-ve= 34.8 ± 5.1%, P<0.05). This was due to an accumulation of the cells in early stages of erythroid differentiation, with increased proportions of proerythroblasts (GFP+ve: 36.6 ± 7.6%; GFP-ve: 8.6 ± 1.3% P<0.002) and basophilic erythroblasts (GFP+ve: 34.3 ± 3.4%; GFP-ve: 13.2 ± 3.5%, P<0.001) in muHoxa1 Ter119+ cells. The block in erythroid differentiation was also accompanied by significant alterations in the proportions of immature progenitors in recipients of muHoxa1 BM. GFP+ve cells were detectable in HSC and myeloid progenitor cell subsets, however, there was a significant increase in the proportion of megakaryocyte erythroid progenitors (MEPs) within the lineage negative, c-kit+, Sca-1 negative progenitor cell fraction (GFP+ve: 42.9 ± 2.1%; GFP-ve: 8.2 ± 1.3%, P<0.01). Significantly reduced GATA-1 expression was observed in both the GFP+ve proerythroblasts (400-fold) and MEPs (100-fold) compared to GFP-ve populations sorted from the same mice (P<0.001). Taken together, these results suggest that overexpression of FL-Hoxa1 in the absence of Hoxa1-T results in the development of MDS. This is partly due to an accumulation of MEPs and impaired differentiation of erythrocytes, both of which have significantly downregulated expression of GATA-1. Given the striking similarities in hematological phenotype to human patients with MDS, this novel mouse model will be invaluable in identifying the mechanisms contributing to this disease. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4373-4373
Author(s):  
Yuchen Zhang ◽  
Mingfeng Zhao ◽  
Deguan Li ◽  
Xiaoli Cao ◽  
Jie Chen ◽  
...  

Abstract Iron overload (IO) is a pathological phenomenon characterized by excessive iron depositing in tissue and caused by hereditary hemochromatosis or repeated blood transfusions in some diseases such as beta thalassemia, MDS. IO can induce organ dysfunctions, such as hepatic fibrosis, diabetes and cardiac diseases and even hematopoiesis suppression. Bone marrow-derived mesenchymal cells (BM-MCs), which located in the hematopoietic niche, functions as maintaining the stability of the hematopoietic microenvironment and supporting hematopoiesis via secreting many cellular factors and differentiating into other stromal cells. Our preliminary researches suggest that IO may induce murine hematopoietic cells injury and suppress hematopoiesis of IO patients. (Lu et al., Eur J Haematol, 2013; Chai et al., blood, 2013 abstract). Here we observed the inhibitory effects of IO onthe BM-MCs and preliminarily discussed the related mechanism. Mice were divided into four equal groups and designed as non-treated control mice group (control): mice were treated with isovolumic saline by intraperitoneal injection everyday. IO mice group (IO): mice were treated with 25mg/ml iron dextran by intraperitoneal injection every 3 days. The iron-chelation mice group (Deferasirox, DFX): IO mice were treated with 125mg/kg DFX by gavage everyday. Anti-oxidative group: IO mice were treated with 40 mM N-acetyl-cysteine (NAC, anti-oxidative regent) in drinking water. All experiments were carried at the same time for 4 weeks. Then BM-MCs were isolated from compact bone at the end of forth week according to the reference (Zhu H et al, Nature Protocol, 2010). Iron deposits in BM-MCs were observed by morphological study and labile iron pool (LIP) level of BM-MCs was detected using the calcein-AM method. The results suggest that the iron deposits and LIP level of BM-MCs in IO mice was significantly increased, which could be partly alleviated by DFX (p<0.05). Then, the biological characteristic and supporting function on normal bone marrow mononuclear cells (BMMNCs) of BM-MCs were evaluated. Firstly, the proliferation ability of BM-MCs was assessed by double time (DT) and cell counting kit-8(CCK8) assay. BM-MCs of IO mice showed a longer DT (2.07±0.14)d compared to that of control group (1.03±0.07)d, which could be partly reversed by DFX (1.52±0.07)d and NAC (1.68±0.03)d (P<0.05),respectively. The similar results were obtained by CCK8 assay,too. Secondly, the osteoblastic differentiation ability of BM-MCs was evaluated via ALP staining and Alizarin Red S staining, as well as osteogenic gene expression. IO inhibited the ALP expression and mineralized nodules formation of osteoblast. Meanwhile, we observed a significant decrease in ALP and RUNX2 mRNA expression by RT-PCR, which also can be reversed by iron-chelation and anti- oxidative therapy. However, Oil red O (ORO) staining indicated that lipid accumulation notablely increased in IO group lipoblast compared with control group, which meaned that IO increased adipogenic differentiation of BM-MCs. Thirdly, the supporting function of IO BM-MCs on normal BMMNCs is assessed by the colony-forming unit (CFU) assay. Normal control or IO BM-MCs were co-cultured with the same pool of normal BMMNCs, After 7 days, co-cultures containing normal BM-MCs formed more CFU than that of control group. These results suggest that the proliferation potential and the differentiation potential of BM-MCs were decreased in the IO environment. Further more, we also explored the possible mechanism of this phenomenon. Our results showed a marked increase in reactive oxidative species (ROS) level of IO group than that of control group, which could be reversed by NAC and DFX treatments, respectively (P<0.05). Quantitative RT-PCR analysis of genes associated with ROS were performed, the results showed that FOXO3 decreased in IO group compared with control group (p<0.01). However there was no significant difference in PI3K expression between IO group and control group. All above implied that FOXO3 may play an important role in IO catalyzed oxidative stress. In conclution, dysfunction of BM-MCs, an important component of hematopoietic microenvironment, may play a crucial role in the suppression of hematopoiesis during IO. Improving the function of BM-MCs may serve as a new strategy to enhance normal hematopoiesis in IO bone marrow . Disclosures No relevant conflicts of interest to declare.


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