Hepcidin as a Therapeutic Tool to Limit Iron Overload and Improve Anemia In β-Thalassemia

Abstract 1009

The principal regulator of iron homeostasis, the hepatic peptide hepcidin (Hamp), degrades the iron-transport protein ferroportin (Fpn) localized on absorptive enterocytes, hepatocytes and macrophages. Low Hamp expression has been associated with iron overload in patients and mice affected by β-thalassemia intermedia (th3/+). Our hypothesis is that more iron is absorbed than required for erythropoiesis in β-thalassemia. Therefore, we propose that limiting the dietary iron intake of th3/+ mice either by feeding them a low iron diet or increasing their Hamp expression will have a beneficial effect on iron overload with no effects on erythropoiesis. In particular, since Hamp expression is low in β-thalassemia, a moderate increase of Hamp expression should not interfere with erythropoiesis by preventing release of iron from macrophages. However, we predict that very high levels of Hamp expression will limit the recycling of iron from macrophages, thereby exacerbating the anemia. We first analyzed wt and th3/+ mice fed diets containing a physiological amount of iron (35 ppm) or low iron (2.5 ppm) for 1 and 5 months. These mice were then compared to wt and th3/+ mice expressing a transgenic Hamp (THamp and THamp/th3, respectively). In wt mice, the low-iron diet decreased tissue iron levels leading to anemia (Hb: 14.6±0.7 g/dL and 8.6±2.4 g/dL at 1 and 5 months, respectively). In th3/+ mice fed the low-iron diet, the amount of iron in the liver and spleen decreased over time and after 5 months was 10 times lower than at the beginning of treatment. However, in this case the low-iron diet did not worsen the anemia, (Hb: 8.2±1.3 g/dL vs. 7.8±1.8 g/dL at 1 and 5 months, respectively). In the case of THamp and THamp/th3 mice, we stratified those animals whose transgenic Hamp expression was moderate (2-4 higher) or high (>4 times higher) compared to the endogenous Hamp expression in control mice. In THamp animals expressing a moderate level of Hamp, the total iron content of the liver was decreased (65±21 μg vs. 131±31 μg in wt controls) while no significant changes were detected in the spleen. THamp mice also exhibited anemia (Hb: 11.2±1.8 g/dL vs. 13.9±1.1 g/dL at 1 month). The iron content of the liver and spleen was reduced in THamp/th3 (127±86 μg vs. 234±49 μg and 131±88 μg vs. 271±74 μg, respectively, compared to th3/+ controls), while their hematological values were dramatically improved. Splenomegaly was also significantly reduced. Similar findings were observed at 5 months. Looking at animals expressing high levels of transgenic Hamp, both THamp and THamp/th3 mice exhibited vast accumulations of iron in macrophages, profound anemia, reticulocytosis and increased splenomegaly, confirming that high levels of Hamp block iron recycling and are detrimental to erythropoiesis. Interestingly, in THamp/th3 mice expressing a moderate level of Hamp we observed that the increase in hemoglobin levels was associated with increased red cell numbers but reduced mean corpuscular hemoglobin levels. Paradoxically, this could indicate that reduction of the anemia in THamp/th3 mice is mediated by decreased heme synthesis. α-Globin/heme aggregates lead to ineffective erythropoiesis and a limited red cell life span by producing reactive oxygen species and altering the structure of red cell membranes. Compared to th3/+ mice, THamp/th3 mice exhibited reduced heme contents, insoluble membrane-bound α-globins and reactive oxygen species resulting in an increased life span and more normal morphology of their red blood cells. While the number of red blood cells was increased, the number of reticulocytes, and the total number of erythroid precursors in the spleen were reduced. This was associated with a reduction in reactive oxygen species. Cell cycle analysis of the erythroid cells at different stages of differentiation, expression of heme related proteins and synthesis of α- and β-globin chains in THamp/th3 mice is in progress. Overall, this study indicates that use of hepcidin might be effective in reducing iron overload and improving erythropoiesis in β-thalassemia thereby limiting toxicity due to heme not incorporated into the adult hemoglobin tetramer. In conclusion, we believe this study provides the first evidence that hepcidin could be utilized for the treatment of abnormal iron absorption in β-thalassemia and other related disorders, with additional beneficial effects on ineffective erythropoiesis, splenomegaly and anemia.