Ella Guy

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Stem Cell Transplantation Reveals That Absence of Macrophage CD36 Is Protective Against Atherosclerosis

Objective—CD36 is expressed on multiple cell types and has numerous functions, a subset of which can impact on atherogenesis. In previous work, we demonstrated that CD36 absence was protective against lesion formation. The current objective was to determine whether absence of macrophage CD36 alone was protective. Methods and Results—Lethal irradiation and stem cell transfer were used to create chimeric mice that did or did not express macrophage CD36 in the context of the Apo E-null model of atherosclerosis. After engraftment, mice were fed a Western diet for 12 weeks. White cell counts, plasma levels of lipoproteins, triacylglycerol, and nonesterified fatty acids were determined, and glucose tolerance tests were preformed. Lesion area was assessed quantitatively after oil red O staining. Mice lacking CD36 in macrophages alone were profoundly protected against atherosclerosis (88.1% reduction of lesion area throughout the aortic tree). Re-introduction of macrophage CD36 resulted in a 2.11-fold increase in lesion area. There were no differences in engraftment, macrophage recruitment, glucose tolerance, weight, and total, low-density lipoprotein, and high-density lipoprotein cholesterol among the groups. Lesions contained similar percent macrophage antigen-positive area. Conclusion—Protection in this model is primarily caused by loss of CD36 macrophage function.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36

Fatty acid translocase (FAT)/CD36 has been associated with diverse normal and pathologic processes. These include scavenger receptor functions (uptake of apoptotic cells and modified lipid), lipid metabolism and fatty acid transport, adhesion, angiogenesis, modulation of inflammation, transforming growth factor-β activation, atherosclerosis, diabetes and cardiomyopathy. Although CD36 was identified more than 25 years ago, it is only with the advent of recent genetic technology that in vivo evidence has emerged for its physiologic and pathologic relevance. As these in vivo studies are expanded, we will gain further insight into the mechanism(s) by which CD36 transmits a cellular signal, and this will allow the design of specific therapeutics that impact on a particular function of CD36.

Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

Dietary Cholesterol Plays a Role in CD36-Mediated Atherogenesis in LDLR-Knockout Mice

Objective—CD36 has been shown to play a role in atherosclerosis in the apolipoprotein E–knockout (apoEo) mouse. We observed no difference in aortic lesion area between Western diet (WD)–fed LDLRo and LDLRo/CD36o mice. The objective was to understand the mechanism of CD36-dependent atherogenesis. Methods and Results—ApoEo mice transplanted with bone marrow from LDLRo/CD36o mice had significantly less aortic lesion compared with those transplanted with LDLRo marrow. Reciprocal macrophage transfer into hyperlipidemic apoEo and LDLRo animals showed that foam cell formation induced by in vivo modified lipoproteins was dependent on the lipoprotein, not macrophage type. LDLRo and LDLRo/CD36o mice were fed a cholesterol-enriched diet (HC), and we observed significant lesion inhibition in LDLRo/CD36o mice. LDL/plasma isolated from HC-fed LDLRo mice induced significantly greater jnk phosphorylation, cytokine release, and reactive oxygen species secretion than LDL/plasma from WD-fed LDLRo mice, and this was CD36-dependent. HC-fed LDLRo mice had higher circulating levels of cytokines than WD-fed mice. Conclusions—These data support the hypothesis that CD36-dependent atherogenesis is contingent on a proinflammatory milieu that promotes the creation of specific CD36 ligands, not solely hypercholesterolemia, and may explain the greater degree/accelerated rate of atherosclerosis observed in syndromes associated with inflammatory risk.

Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in β-thalassemia

Abstract: To develop new treatments for β‐thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp‐knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of β‐thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.