Increased Intracellular Concentration of Reactive Oxygen Species Mediated the Deficient Hematopoiesis of Iron Overload Bone Marrow

Abstract

4247 Iron overload is caused by multiple blood transfusion and excess gastrointestinal absorption, leading to most of the mortality and morbidity associated with anemia diseases (i.e. aplastic anemia, myelodysplastic syndromes, thalassemia and myelofibrosis). It can cause tissue damage and ultimately dysfunction of visceral organs (mainly in the heart, liver, and endocrine glands). Nevertheless, it is unknown whether iron overload affects the hematopoiesis of bone marrow (BM). In recent years, a number of papers reported that iron chelation therapy could enhance the erythropoiesis and even reduce the cytopenia in iron overload anemia (i.e. myelodysplastic syndromes and myelofibrosis). As it is well known that iron overload could increase the production of reactive oxygen species (ROS) in the tissue, and also ROS could affect the hematopoiesis of BM, we hypothesized that iron overload could increase the ROS of BM, and then result in the deficient hematopoiesis. To confirm this hypothesis, we studied the relationship between ROS and the hematopoiesis of iron overload BM. The intracellular concentration of ROS in cells were analyzed by a flow cytometer after incubated with 50 μM (final concentration) 2′–7′-dichlorofluorescin diacetate (DCF; Sigma) for 10 min at 37°C in a humidified atmosphere of 5% CO2 in air. We found that the ROS in the hematopoietic cells of BM from 26 patients with iron overload (16 cases with myelodysplastic syndromes and 10 cases with myelofibrosis) was much higher than that of normal control (p<0.05), which in accord with the lower production of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) in patients with iron overload. After the patients received iron chelation therapy with deferasirox for more than 6 months, the ROS decreased distinctly and the hematopoietic colony forming recovered subsequently. As a result, 20 cases (76.9%) reduced blood transfusion, 12 cases (46.2%) increased neutrophil counting, 7 cases (26.9%) increased platelet counting, and 4 cases (15.4%) reduced the cytopenia. In vitro, we set up an iron overload model by adding ferric citrate into the mononuclear cells from normal BM and culturing for 24 hours, and confirmed the model by the detecting of labile plasma iron. We found that the concentration of ROS in iron overload BM was increased for 3.5 folds in erythroid cells, 2.1 folds in granulocytes and 1.3 folds in CD34+ cells, respectively. Again, the hematopoietic colony forming in iron overload BM was much lower than that of normal control. Notely, the number of CD34+ cells was decreased about 45% in iron overload BM. When treated the hematopoietic cells from iron overload BM with deferasirox or/and antioxidants (N-acetyl-L-cysteine), the hematopoietic colony forming was recovered in accord with the decreasing of ROS. Furthermore, the apoptosis of hematopoietic cells from iron overload BM was much higher than that of normal control detecting with PI-Annexin V double-stainning method. And also, detecting with western-blot method, we found the ATM kinase was down-regulated and its downstream factor, p38MAPK was notably actived in the hematopoietic cells from iron overload BM, which indicated that ROS related signal pathway was involved in the deficient hematopoiesis of iron overload BM. In sum, our study confirmed that increased intracellular concentration of ROS mediated the deficient hematopoiesis of iron overload BM for the first time. Further study on the mechanism would be helpful to find the target (for example, antoxidant treatment) and improve the efficacy on the treatment of ineffective hematopoiesis in patients with iron overload. Disclosures: No relevant conflicts of interest to declare.

Free iron catalyzes oxidative damage to hematopoietic cells/mesenchymal stem cellsin vitroand suppresses hematopoiesis in iron overload patients

“…A recent study revealed that the suppressed erythroid differentiation induced by iron overload was mediated by ROS and could be restored by iron chelator or antioxidant (14). Our preliminary study also proved that iron overload affected the function of hematopoietic stem/progenitor cells by the production of ROS (15). Although these findings may partly explain how iron overload affects the hematopoiesis, there is still no answer to the question whether hematopoietic environment is affected during the process and whether ROS-related signaling pathway participates in this process.…”

Impact of labile plasma iron and iron chelation on the viability of cultured mononuclear cells from patients undergoing autologous hematopoietic stem cell transplantation

“…In fact, iron overload in the bone marrow has been shown to increase ROS concentrations in erythroid cells, granulocytes, and CD34+ cells, and this was associated with decreased hematopoietic colony formation relative to that in normal bone marrow. These findings confirmed that ROS signaling played a role in deficient hematopoiesis in iron-overloaded bone marrow [ 8 ]. Another in vitro study showed that free iron inhibited colony formation by erythroid and granulocyte-macrophage progenitors and reduced the size of colonies in a dose-dependent manner [ 9 ].…”

Effects of Iron Overload on the Bone Marrow Microenvironment in Mice

“…It is known that iron is the main catalyst of ROS in an organism, and a growing body of evidence demonstrates that there is a positive correlation between ROS levels and LIP [ 12 , 13 ]. This finding prompted examination of whether IO induced oxidative stress in BM-MSCs in vivo.…”

“…Preliminary research revealed that under iron overload conditions, MSCs were deficient in proliferation and exhibited increased apoptosis. This progressed relative to catalyzing oxidative stress, and there was a positive correlation between ROS levels and labile iron pool (LIP) [ 12 , 13 ]. It has been known that the effect of IO on hematopoietic stem/progenitor cells involved cellular senescence and apoptosis by up-regulating ROS level [ 14 , 15 ].…”