Elizabeta Nemeth, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This proposal outlines a three year training plan for transition to an independent investigator. During her previous studies in the laboratory of Dr. Tomas Ganz at the UCLA, the applicant developed a background in iron metabolism and acquired extensive related research skills. She will now use the mentored period to fully develop her own research ideas, learn laboratory management and strategic planning skills, and obtain independent funding to establish her own laboratory. She will also broaden her education in research ethics. Tomas Ganz will serve as the main scientific mentor. A pioneer in innate immunity and iron metabolism, Dr. Ganz combines impressive scientific and medical knowledge with superb teaching and management skills. The training plan also involves mentorship from Dr. Jerry Kaplan, one of the most prominent investigators and mentors in the field of iron metabolism, and Dr. Sabeeha Merchant, an expert on bacterial and plant iron metabolism who will also provide guidance in formal teaching and academic career development. [unreadable] Hepcidin is the key iron-regulatory hormone and the pathogenic factor in a spectrum of iron disorders. The proposed research project focuses on how hepcidin is regulated by iron and hypoxia. This is perhaps the most important question in the field of iron metabolism today. Specifically, we will: [unreadable] 1. Examine the regulation of hepcidin synthesis by iron [unreadable] 1a. Identify the hemojuvelin receptor(s) [unreadable] 1b. Analyze possible interactions of hemojuvelin with TfR2 and HFE [unreadable] 1c. Examine whether an extrahepatic iron sensor modulates hepcidin synthesis via soluble hemojuvelin [unreadable] 1d. Identify the elements of the hepcidin promoter needed for the response to hemojuvelin [unreadable] 2. Examine the regulation of hepcidin synthesis by oxygen [unreadable] 2a. Determine how HIF is involved in hepcidin regulation by oxygen [unreadable] 2b. Identify the elements of hepcidin promoter necessary for the response to hypoxia [unreadable] Defining the molecular pathways of hepcidin regulation by iron and oxygen is essential for the understanding of systemic iron homeostasis as well as the pathogenesis of iron overload diseases, and may provide the foundation for design of new agents for the diagnosis and treatment of these disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Anemia of inflammation (AI, also called anemia of chronic disease) is associated with a wide variety of infectious and inflammatory conditions and contributes to their morbidity. The pathogenesis of AI is incompletely understood and treatment options are limited. Erythropoietin, intravenous iron or their combination are increasingly used to treat this condition but the therapy is only partially effective and frequently requires high doses of these agents with increasing risk of side effects. Thus new, more specific therapeutic modalities are greatly needed. The significance and timeliness of this area was recognized by a recent RFA (PAS-08-019). Hepcidin is the key regulator of systemic iron homeostasis and an acute phase reactant. Recent studies indicate that increased hepcidin production in inflammatory conditions is a principal contributor to the development of AI and is a likely cause of resistance to erythropoietin. Therefore, antagonizing hepcidin activity is a promising approach for improving therapies for AI. However, the specific molecular pathways that mediate the effect of hepcidin on ferroportin are poorly understood. We propose to define comprehensively the pathways activated by the interaction of hepcidin with its receptor ferroportin and identify compounds that antagonize these pathways. Specifically, we will: 1) Systematically discover hepcidin antagonists by high-throughput screening (HTS) with small molecule inhibitors and siRNA 2) Using identified hepcidin antagonists, characterize Fpn internalization pathways in cellular models 3) In animal models of AI, define the efficacy of selected antagonists and the contribution of the hepcidin- ferroportin axis to the disease process. The proposed approach will answer fundamental questions about the pathogenesis of AI and the related problem of resistance to erythropoiesis-stimulating agents. It may also provide lead compounds for further development as therapeutics. In the aggregate, this proposal effectively and comprehensively addresses an important and timely scientific and medical problem. PUBLIS HEALTH RELEVANCE: The proposed project will help understand and treat a common form of anemia which develops in infections and inflammatory disorders.

ABSTRACT The possible role of iron in the promotion of atherosclerosis is a major unresolved question. Various studies in animal models and humans over the last 30 years assessed the effect of increased body iron on atherosclerosis but have yielded inconsistent results. In the last decade, our understanding of iron biology underwent a radical revision, raising questions about the design and interpretation of numerous studies on the subject. We will use the new understanding of iron homeostasis to explore the role of iron in atherosclerosis. Research focused on cardiovascular disease and nutrition areas with inconclusive evidence of benefit or risk has been identified as a high priority as indicated in a recent NIH initiative (PA-09-244). We propose to test the following conceptual framework. Iron, known as a potent catalyst for generation of reactive oxygen species, likely accelerates atherosclerosis by increasing oxidative stress in the plaque, oxidizing accumulated lipids and promoting inflammation. In atherosclerosis, as in other inflammatory diseases, systemic and local inflammation increases the production of the iron-regulatory peptide hormone hepcidin. Hepcidin functions by inhibiting the release of iron from macrophages, and would have the same effect in the atherosclerotic plaque on macrophages that ingest erythrocytes and apoptotic/necrotic cells. The hepcidin-mediated accumulation of iron in plaque macrophages and the resulting inflammation constitutes a self-amplifying process and is an important promoter of atherosclerosis. Our specific aims are: 1. Define the effect of atherosclerosis on systemic (hepatic) and local (plaque macrophage) hepcidin production in apoE-/- mice 2. Define the effect of increased macrophage iron on atherosclerosis progression in flatiron mice on apoE-/- background Successful completion of this study will help resolve important questions about the role of iron in atherosclerosis. Similar to other modifiable risk factors, strategies for reduction of plaque iron could be devised to help reduce the morbidity and mortality associated with cardiovascular disease.

? DESCRIPTION (provided by applicant): The iron transporter ferroportin is critical for delivering iron into plasma from duodenal enterocytes absorbing dietary iron, macrophages recycling old red blood cells, and hepatocytes storing iron. The interaction of ferroportin with its ligand, the hepatic hormone hepcidin, is the key event in systemic iron homeostasis. After it binds hepcidin, ferroportin is degraded, thereby limiting iron entry into plasma. Dysregulation of the hepcidin- ferroportin axis underlies most common iron disorders, including anemia of inflammation, anemia of chronic kidney disease, anemia of cancer, hereditary hemochromatosis and iron-loading anemias. Ferroportin is the only known cellular iron exporter in vertebrates and is conserved down to invertebrates and plants. Despite its obvious biological importance, very little is known about the ferroportin structure and the mechanisms by which ferroportin transports iron. In a recent breakthrough, we identified the prokaryotic ortholog of Fpn and obtained its structure by X-ray crystallography. We are now poised to make rapid progress toward complete understanding of the structural basis of Fpn function by combining X-ray crystallography of mammalian Fpn with detailed structure-guided mutational and functional analyses of metal transport and hepcidin-ferroportin interaction in mammalian cells and Xenopus oocytes. Our Specific Aims are: Aim 1. Determine the structure of ferroportin by X-ray crystallography. While these efforts are ongoing, we will use the structure of prokaryotic Fpn ortholog to guide further studies of the transport mechanism, specific function of conserved residues, and as a framework for functional and mutagenesis work on the higher orthologs. Aim 2. Determine the mechanism of iron transport by ferroportin. These studies will determine the driving forces, ion coupling, and calcium gating of Fpn-mediated iron transport. Aim 3. Discover the structural determinants of ferroportin function and malfunction. We will use structure- and human disease guided mutagenesis to probe critical residues involved in iron binding and translocation, transporter gating, pH dependence, oligomerization, and hepcidin binding. Successful completion of the proposed studies is of fundamental importance for iron biology. Its significance also extends to general biology: identifying the structure of a new class of membrane transporters and defining the mechanism of iron transport will impact studies of other metal and ion transporters. Finally, understanding Fpn iron-transporting function and its regulation by hepcidin is biomedically significant. The proposed studies will generate much more definitive mechanistic and structural insights which will guide the development of improved small and large molecule therapeutics for iron-restrictive anemias and iron overload disorders.

PROJECT SUMMARY Adequate iron availability during pregnancy is essential for fetal development and maternal health. Iron deficiency and its most common manifestation, anemia, are highly prevalent during pregnancy and can have adverse effects on the mother and the fetus. Recognition of detrimental effects of iron deficiency has led to the policy of universal iron supplementation in many countries including the US. However, in developed countries, more pregnant women are iron-replete than iron-deficient. Indiscriminate iron supplementation in this setting may be not only unnecessary, but may even have harmful effects related to increased oxidative stress or potential adverse interaction with inflammation. Despite its importance, little is known about the basic physiology of iron regulation during pregnancy, or how it is altered in complicated pregnancies. Specific Aim 1. We will define the role of maternal and fetal hepcidin in regulating iron homeostasis during pregnancy. Our preliminary data in mouse models indicate that maternal iron-regulatory hormone hepcidin must be suppressed during pregnancy to ensure sufficient iron availability for placental transfer, and that trophoblast is an important source of the hepcidin-suppressive activity. We will identify the mechanism(s) by which maternal hepcidin is suppressed in healthy pregnancy; and determine whether maternal hepcidin levels are inappropriately increased in human inflamed pregnancies to promote maternal iron restriction. Specific Aim 2. Our preliminary data show that during maternal iron deficiency or excess, placental iron transporters are regulated to ensure the maintenance of placental iron homeostasis. In response to maternal iron deficiency in both mice and humans, this mechanism sequesters iron in the placenta at the expense of providing adequate iron to the fetus, a phenomenon we termed the ?selfish placenta?. This has important implications for understanding the pathogenesis of fetal iron deficiency. We will define the regulatory circuitries and biological relevance of the selfish placental response in pregnancy. Specific Aim 3. Our preliminary data in mouse models demonstrate a dramatic adverse interaction between maternal iron excess and maternal inflammation during pregnancy, targeting placental endothelium, and resulting in fetal malformations and embryonic lethality. We will define the underlying mechanisms by focusing on maternal inflammation and pregnancy hormones, as well as placental and fetal inflammation, oxidative stress, and cell death pathways. Our proposal will answer fundamental questions about the pathophysiology of maternal and fetal iron regulation during pregnancy. Long-term, it has the potential to change our approaches to managing iron disorders during pregnancy.

Adequate iron availability during pregnancy is essential for fetal development and maternal health. Iron deficiency and its most common manifestation, anemia, are highly prevalent during pregnancy and can have adverse effects on the mother and the fetus. Systemic maternal inflammation during pregnancy is also quite common, and either results from infections (bacterial, viral, parasitic), or accompanies chronic conditions including obesity, diabetes, and autoimmune diseases. Adverse outcomes associated with inflamed pregnancies include spontaneous abortion, preterm labor, intrauterine growth restriction, fetal death, gross developmental abnormalities and autistic behaviors in offspring. Our preliminary studies in mice demonstrated a surprising synergistic adverse interaction between maternal iron deficiency and inflammation that led to embryotoxicity which was not observed with either condition alone. Compared to dams with normal iron status, those that were iron-deficient had significantly higher frequency of embryo abnormalities in the setting of acute maternal inflammation caused by LPS injection. We aim to define the underlying mechanisms of this synergy: Aim 1. Determine the effect of iron deficiency on maternal, placental and embryo inflammation. Using our mouse models of LPS-triggered inflammation, we will examine whether iron deficiency in pregnancy enhances pro-inflammatory response or alters immune cell census in the mother, placenta and embryo. Aim 2. Characterize the iron-dependent regulation of TNF receptor 1 Based on the strong correlation between placental TNFR1 and TFR1 in both humans and mice, we hypothesize that cellular iron status regulates TNFR1 levels. We will define the specific placental cell type that increases TNFR1 in response to iron deficiency, and examine candidate mechanisms regulating TNFR1. . Aim 3. Determine the role of the TNF pathway in adverse synergy in vivo?Our preliminary data implicated TNF?-TNFR pathway in mediating adverse outcomes in iron-deficient inflamed pregnancies. We will use mice with endothelial- or trophoblast-specific deficiency of TNFR1 to determine whether the TNF pathway is required for the adverse synergy in the model of acute inflammation. Aim 4. Define the effect of iron deficiency on placental cellular transcriptome. We will complement our candidate-driven approaches with an unbiased single-cell RNAseq on mouse placentas from iron-deficient and iron-replete pregnancies treated with LPS to gain detailed insight into how iron deficiency alters placental cell populations as well as their transcriptional activity in response to inflammation. Our proposal analyzes a novel interaction between iron deficiency and inflammation. Considering the high global prevalence of this combination of disorders in pregnant women, the subject has outstanding translational importance, and our studies could help explain commonly observed adverse outcomes. Long-term, our findings have the potential to influence the management of iron and inflammatory disorders of pregnancy.