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.