RoFAR awards an additional 1.2 million Swiss Francs to fund ground-breaking anaemia research in Germany, Israel and the USA

The award winners with their institutions and a description of the projects are:

Prof. M. Amin Arnaout Massachusetts General Hospital, Charlestown, USA   Role of the zinc finger transcription factor ZBP-89 in haematopoiesis

Haematopoietic stem cells (HSCs) sustain the production of blood cells throughout the lifetime of an individual. HSCs also possess the capacity for inducting immune tolerance to engrafted solid organs (such as the kidney) when combined with HSC transplantation from the same donor. Despite the therapeutic benefits of being able to increase the number of HSCs as blood-forming and immune-modulatory cells, the key factors that control formation of HSCs during embryonic/foetal development and regulate their sustained capacity to generate all blood elements in the adult remain incompletely understood.

We have identified the DNA-binding protein ZBP-89 as master regulator of haematopoiesis during embryonic life in zebra fish, as well as in murine embryonic stem cell cultures differentiated into HSC subsets in vitro. In preliminary studies, we also found that newborn mice that are homozygous for a hypomorphic ZBP-89 allele die within 1-2 days after birth with severe anaemia, reflecting a critical role for ZBP-89 in foetal erythropoiesis. We now propose to: 1) examine the requirement for ZBP-89 in foetal as well as adult haematopoiesis using engineered mice in which ZBP-89 is temporally and selectively inactivated in haematopoietic tissues; 2) assess the effects of ZBP-89 over-expression in bone narrow-derived HSCs on blood lineage commitment in vivo as well as in vitro.

The results from these studies will define the role of a previously unrecognised master regulator of blood cell development, and will likely provide novel approaches for expanding the use of bone marrow stem cells for gene transfer and bone marrow reconstitution. The additional role of ZBP-89 as a key regulator of foetal erythropoiesis may also provide new ways of enriching the red blood cell population from less differentiated precursors to treat anaemia.

Dr David M. Briscoe Children’s Hospital Boston, USA   Erythropoietin and vascular endothelial cells

 Erythropoietin (EPO), as its name suggests stimulates the proliferation and differentiation of erythroid (red blood cell) lineage progenitor cells. It is widely used as a treatment for anaemia in several chronic disease states including chronic renal disease. However, a growing body of evidence indicates that EPO has effects beyond the correction of anaemia. In addition, while it has been known for some time that vascular endothelial cells (EC) lining blood vessels express EPO receptors, only recently has the effect of EPO on EC been studied. Following inflammatory insults (where vascular EC are the targets of injury), damaged cells slough into the circulation. Replacement of EC occurs via the proliferation of neighbouring EC and/or recruitment of endothelial progenitor cells (EPCs) from the circulation. In the absence of microvascular repair, nutrient and oxygen deprivation to cells results in cellular dysfunction and ultimately in tissue death. Protection and/or repair of the vasculature have the potential to prevent cellular injury. To our knowledge, little is reported on the effects of EPO on human EC, and no study has evaluated its biology in human EPC. In this research proposal, we plan to identify the effects of EPO on human EPC and mature human EC in established in vitro models. We will assess the effect of EPO on angiogenic as well as protective intracellular signalling in EC, and we will determine its effect on the maintenance of vascular integrity in vivo. The ability of EPO to interact with vascular EC suggests that it has the potential to be a novel agent to induce microvascular protection and vascular repair before (such as in kidney organ donors) or following tissue injury (such as in acute renal injury). Also, understanding this biology is likely to give insight into the potential pro-angiogenic, pro-tumourigenic effects of EPO in humans.

Dr Saghi Ghaffari Mount Sinai School of Medicine, New York, USA   Foxo3 regulation of erythropoiesis

In red blood cells, haemoglobin carries oxygen and is therefore essential for life. However, the process of haemoglobin formation and its interaction with oxygen are a source of production of oxygen radicals that are toxic for red blood cells. We have identified a nuclear factor (Foxo3) that detoxifies oxygen radicals in red blood cells, and through this process, extends the lifespan of these cells. Experiments described here build upon findings in our laboratory suggesting that this regulatory factor may control the rate of red blood cell formation by modulating the levels of oxygen radicals. In our research, we will use both red blood cell precursors derived from genetically modified animals and from human embryonic stem cells to investigate how Foxo3 modulates genes involved in oxygen radical detoxification. We will identify additional genes that are regulated by Foxo3 and factors that, together with Foxo3, control the rate of red blood cell formation. These factors may provide new targets for the treatment of anaemia.

Dr Esther Meyron-Holtz Technion – Israel Institute of Technology, Haifa, Israel   Macrophage’s reaction to erythrophagocytosis

Macrophages (MPs) are a heterogeneous family of phagocytosing cells which play a major role in growth and development, the immune system and systemic iron recycling in mammals. Phagocytosis of pathogens initiates a range of pro-inflammatory responses, while phagocytosis of apoptotic cells induces anti-inflammatory responses. Many factors such as receptor recognition and substances released from the phagocytosed particle determine the signal transduction and response of the MP to phagocytosis.

Senescent red blood cells (sRBCs), similar to pathogens and apoptotic cells, are recognised and phagocytosed by MPs, a process called erythrophagocytosis (EPC). EPC is a regular activity of MPs that eliminates about 3 million RBCs per second in humans. However, neither an inflammatory nor an anti-inflammatory response of MPs to physiological EPC has been elucidated.

Our research will focus on the molecular characterisation of the MP response to EPC. We hypothesise that both the MP receptors involved in the recognition of sRBCs, and the large amount of haem and its breakdown products released from decaying sRBC, may play a role in determining the MP response to EPC.

Specific aims:

1.  Characterisation of cellular events following sRBC phagocytosis and breakdown.

Approach: MPs before and after EPC of aged RBCs will be analysed in vivo. Changes in MP activation status at different stages of haem catabolism will be characterised by analysing candidate cytokines and by microarray.

2.  Identification of the receptors and additional factors, such as haem breakdown products involved in the signal transduction induced by physiological EPC.

Approach: Specific blocking of MP receptors, receptor deletions with RNAi and genetic deletions will be used to identify the key players in the MP response to EPC.

A decreased RBC lifespan and changes in RBC surface markers are reported in diabetes mellitus, haemoglobinopathies and other conditions. This implies that these pathologies may change MP activation status and immune homeostasis. Future studies will include the effect of this response on systemic iron recycling and the immune system.

Prof. Heike L. Pahl University Hospital Freiburg, Germany   Role of transcription factor NF-E2 in mediating anaemia and erythrocytosis

The generation of red blood cells is a continuous process in the body. In a healthy person, millions of red blood cells are produced every day, just as millions are destroyed because they have aged or become used. Since red blood cells are the main cell type in blood, the number of red blood cells in the blood determines the cell/fluid balance of the blood

It is vital that there are sufficient cells in the blood to supply oxygen, since red blood cells carry oxygen to all parts of the body. There are various conditions in which there are too few red blood cells in the blood. These are referred to collectively as “anaemia”. Symptoms of anaemia include fatigue and listlessness, as the body tries to preserve valuable oxygen, which is in short supply. In contrast, if the percentage of red blood cells is too high, the blood becomes viscous, flows more slowly and becomes more likely to form a blood clot. This dangerous condition is known as “polycythaemia” – too many cells in the blood.

Since it is so important for the body to maintain the optimal number of red blood cells in the blood, the production of red blood cells is tightly regulated and controlled. However, to date, this process is not understood at a molecular level. Due to this lack of understanding, it is also unclear what changes take place when the system malfunctions, such as in patients who develop anaemia or polycythaemia.

In this project, we will investigate the role of a single protein, called “transcription factor NF-E2” in regulating red blood cell production. We will determine its role in causing anaemia and polycythaemia in patients affected by a certain group of blood disorders, called “myeloproliferative disorders”. We hope to use our understanding to develop better therapies for these patients.

Prof. Mitchell J. Weiss Children’s Hospital of Philadelphia, USA   Regulation of erythropoiesis by microRNAs

MicroRNAs are a newly discovered class of tiny RNAs that regulate gene expression, tissue development and carcinogenesis.  We discovered a microRNA gene, termed miR144/451, which is important for red blood cell development.  We showed that miR144/451 is conserved in evolution and expressed at high levels specifically in mature red blood cells and their precursors.  In zebra fish, loss of miRNA144/451 function causes severe anaemia.  Now, we will study the functions of miRNA144/451 further by ablating the gene in mice and determining the consequences on blood formation.  We will use these mice, along with methods to manipulate miRNA144/451 expression in cultured cells, to better define the genetic pathways through which this important microRNA gene exerts its effects.  If successful, our studies will illustrate new principles in the basic biology of red blood cell formation and function.  In addition, defining the actions of miRNA144/451 could illustrate molecular pathways that could be manipulated pharmacologically for the treatment of various anaemias.