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Cycle X | | | The award winners with their institutions and a description of the projects are:
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Dr Stephen Archer
University of Chicago, USA
Dysfunction of a mitochondrial oxygen sensor causes normoxic activation of HIF-1a and initiates pulmonary arterial hypertension and polycythaemia in fawn-hooded rats and humans: the Warburg Effect in cardiovascular disease |
Pulmonary arterial hypertension (PAH) is a devastating disease of the lung’s blood vessels that primarily affects young women, causing disability and premature death. Despite modern therapies, 18% of PAH patients die within 1 year of diagnosis. A newly recognized feature of PAH is excessive growth of arterial smooth muscle cells in the lung blood vessels. These rapidly growing cells block the circulation of blood and increase blood pressure in the lungs (pulmonary hypertension). We are investigating the mechanism of PAH in Fawn Hooded Rats (FHR), the only animal that spontaneously develops pulmonary hypertension. Although blood oxygen levels are normal, FHR behave as if they were exposed to low oxygen levels (hypoxia), developing pulmonary hypertension that is associated with high hemoglobin levels and activation of a master hypoxic gene regulator called “hypoxia inducible factor” (HIF-1a). Our research suggests that HIF activation causes overproduction of the hormone erythropoietin (EPO), the normal role of which is to increase production of red blood cells. As a consequence, FHR have increased hemoglobin. However, EPO has another effect, which is harmful. EPO can cause cells to grow too fast and blocks programmed cell death (a process which removes “bad” cells from the body). We explore the possibility that in FHR, HIF-1a increases EPO production and causes cells in the blood vessel walls to grow too much, leading to blockage. In addition to testing this idea as a cause of PAH, we try and interrupt it and create a new treatment for PAH. By inhibiting EPO, we may stop excessive cell growth and reduce pulmonary hypertension. Since the therapies we test involve drugs (theophylline, dichloroacetate) that have been used in people with other conditions (asthma, mitochondrial diseases), a beneficial effect in FHR could spark a clinical trial in human PAH.
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Prof. Margaret Baron
Mount Sinai School of Medicine,
New York, USA
Multispectral imaging cytometry analysis of surface protein redistribution on maturing red blood cells
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Erythroid cells play an essential role in oxygen delivery during embryogenesis and throughout postnatal life. They play vital roles in oxygen delivery and in generating shear forces necessary for normal vasculogenesis. Despite their abundance and indispensable functions, their development and maturation remain poorly understood. We have developed transgenic green fluorescent protein (GFP)-labelled mouse models that permit the clean separation of primitive (EryP) and definitive (EryD) erythroid cells at stages when both lineages are present in the blood. We found that a subset of surface proteins is redistributed towards the extruding nucleus or the emerging reticulocyte as the cells mature. These changes may assist in the engulfment of the extruded nucleus by macrophages or in the release of reticulocytes into the bloodstream. The mechanisms underlying this redistribution are not understood. We propose to capitalise on novel transgenic mouse models developed in our laboratory, in combination with a new and not widely available technology, multispectral imaging cytometry, to study mechanisms underlying terminal maturation and enucleation of erythroid cells. It is anticipated that these studies will lead to the development of better methods for the production of red blood cells for transfusion therapies for anaemias of various aetiologies and may identify novel targets for therapy of Sickle Cell Disease (SCD). The aims of this proposal are (1) To quantify the sub-cellular distribution of alpha4, alpha5 and beta1 integrins, Ter119, and other surface proteins during the maturation of normal embryonic and adult red blood cells; (2) To analyze changes in the cytoskeletal architecture and to correlate these changes with redistribution of cell surface proteins during erythroid cell maturation; (3) To use a transgenic mouse model of SCD to test the hypothesis that Sickle red cells fail to redistribute surface proteins normally.
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Prof. George Daley
Children’s Hospital Boston, USA
Defining the molecular basis of anaemia
in Fanconi Anaemia
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We have developed a unique model system to study the genetics of aplastic anaemia (AA) development in bone marrow failure diseases including Fanconi anaemia (FA). Our system employs RNA-interference against FA genes in human and mouse embryonic stem (ES) cells; the generation of disease-specific lines of induced pluripotent stem (iPS) cells, and the interrogation of functional relevance using in vitro haematopoietic cell derivation. Using this system, we have shown that haematopoiesis is perturbed from the earliest possible stages in FA human cells, but is unimpaired in cognate mouse cells. The commitment to mesoderm in general is not impaired. This enables, for the first time, a large-scale study to identify the genetic interactions between FA genes and haematopoiesis supporting loci in vitro. Our hypothesis is that the functional differences between mouse and human cells in the context of FA will permit the identification of candidate loci that regulate the progression to AA. Furthermore, we are currently generating other lines of disease-specific human iPS cells including from multiple complementation groups of FA, Shwachman-Bodian-Diamond syndrome (SBDS), Diamond-Blackfan anaemia (DBA), Thrombocytopenia Absent Radius (TAR) syndrome, and others. We intend to use these additional iPS lines to complement this project and permit us to draw broad conclusions based upon multiple sources of pluripotent cells and on an even greater variety of genetic backgrounds, and to also include other phenotypically similar conditions that likewise confer a predisposition to the development of anaemia. In addition, as the mechanistic basis for acquired AA is unknown, the study stands to shed light on the pathophysiological underpinnings of acquired, non-FA related AA.
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Dr Emma Derbyshire
North West Food Centre,
Manchester Metropolitan University, UK
Teff (Eragrostis tef) as a functional food for the prevention of pregnancy iron-deficiency anaemia
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It has been estimated that one in two women expecting a baby will be diagnosed with iron deficiency. In turn, iron deficiency can affect the health and well-being, or both, of the mother and child. Studies show that low iron stores prior to conception and low iron intakes during pregnancy may contribute to the problem. Although dietary supplements may be one solution, research indicates that daily compliance is low. Furthermore, prescribed iron supplements may result in uncomfortable side-effects including constipation.
It has been observed in Ethiopia that iron deficiency anaemia is lower than average - a finding that has been attributed to regular "Teff' consumption. Teff (Eragrostis tef) is a staple food usually consumed in the form of Enjera (flat bread prepared using a range of cereals). Research has shown that Teff is a rich source of iron that is easily absorbed by the body.
Although it is believed that regular Teff consumption may prevent the onset of iron deficiency anaemia, there is no research to support this. Therefore, the aim of the present study is to establish whether incorporating Teff in the daily diet may be one way to improve the blood profile and prevent the onset of iron deficiency anaemia in expectant mothers. Study findings will demonstrate whether Teff may be an alternative source of iron that can be easily incorporated into the daily diet of both pregnant mothers and the general public.
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Prof. Agnes Fogo
Vanderbilt University Medical Center,
Nashville, USA
Erythropoietin effects on capillary growth and regression of sclerosis
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Research to slow down the vicious cycle of progressive scarring in chronic kidney disease (CKD) is of paramount importance to avoid dialysis or transplant as the only life-sustaining options. Recently, we have shown in animal models of CKD that existing kidney scarring can be partially reversed when the renin-angiotensin system, a key modulator of hypertension and scarring, is inhibited. Regression of scarring of glomeruli implies both degradation and absorption of the scar tissue, and potentially regeneration and growth of new capillary loops. In this grant, we aim to see if the hormone erythropoietin (EPO) can enhance regression of scarring of glomeruli. EPO is primarily known as a hormone that stimulates red blood cell precursors to develop into mature functional red blood cells. Recently, EPO has been shown to also have effects on other cell types. We will investigate whether EPO augments the growth of glomerular capillary cells, thus promoting repair and restoration of new capillaries. We will investigate whether such effects can occur, using a mouse model of CKD and starting treatment after injury is fully developed, to mirror the human disease setting. We will determine whether EPO has synergy with the main existing treatment used in CKD, drugs that block the renin-angiotensin system, namely angiotensin receptor blocker (ARB). We will study detailed mechanisms in cell culture studies of the key cells in the glomerulus, namely the endothelial capillary cell and the podocyte, which are key for glomerular function. We expect that EPO will have synergistic effects to augment regression of sclerosis, and that this will be accomplished by increasing capillary growth directly, and perhaps also by restoring the health of the podocyte, which is also important for capillary endothelial cell survival.
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Prof. Max Gassmann
Vetsuisse, University of Zurich, Switzerland
EPO's impact on cognitive and learning processes in men and mice
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The “blood hormone” erythropoietin (EPO) fulfills many more functions than just increasing red blood cell number, and the kidney is by far not the only organ that produces EPO. Indeed, cerebral and retinal cells do express this cytokine during impaired tissue oxygenation, a situation physiologically occurring with exposure to high altitude or pathologically with ischemic conditions. It has been shown that EPO application leads to neuroprotective effects in pathological conditions such as stroke in both rodent models and patients. Regarding EPO’s function upon physiological hypoxia, we have described that cerebrally-expressed EPO results in a gender-dependent enhancement of the hypoxic ventilatory response. Thus, EPO links the ventilatory and erythropoietic response to reduced oxygenation.
Nevertheless, we suspect that there are additional physiological functions of hypoxia-induced EPO in the brain. Considering recent publications claiming that elevated EPO levels in the brain of mice and even men can improve memory as well as learning and cognitive tasks, we postulate the following: as oxygen availability declines when reaching higher altitudes, the blood’s oxygenation drops. In turn, this leads to reduced oxygen supply to the brain resulting in poor cognitive, learning and memory performance. To counteract this disadvantageous effect, cerebral cells might express several protective factors, EPO being a prominent one among them. In the proposed experiments, we plan to investigate the impact of hypoxia, and especially of EPO, using a variety of cognitive and learning tasks to test EPO-injected or EPO-overexpressing mice as well as EPO-treated healthy volunteers. In other words, we propose that cerebral EPO has an additional physiological effect, namely to improve the intellectual performance in conditions when the brain “suffers” from reduced oxygen supply. Knowing that at least part of intravenously injected EPO crosses the blood-brain barrier, these observations have a novel clinical implication, especially in neuropsychiatric diseases.
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Dr Iqbal Hamza
University of Maryland, College Park, USA
Functional genomics by genome-wide RNAi of haem homeostasis in animals
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Iron deficiency is the world’s number one nutritional disorder. Over forty years ago, researchers demonstrated that haem, an iron atom enclosed by a porphyrin ring, is absorbed more easily than inorganic iron in the human intestine by an active transporter. However, the protein that transports haem across the intestinal membrane is still unidentified. Even more strikingly, no one has determined how haem is transported and inserted into the globin protein, although haemoglobin was the first protein structure to be elucidated more than fifty years ago. Identifying haem transport pathways has been difficult in eukaryotes, because genetic disruption in the haem biosynthesis pathway results in lethality or pleiotropic phenotypes. Haem is synthesised by a highly regulated multi-step pathway which is conserved throughout evolution. However, we have shown that C. elegans is an exception, because it does not synthesise haem, but rather utilises dietary haem for growth and sustenance. Using C. elegans as an animal model of haem auxotrophy, we identified HRG-1 as the first eukaryotic haem importer. Surprisingly, the C. elegans model system has been relatively unexplored for questions related to haem homeostasis, despite the fact that worms share large numbers of haemoprotein homologs with humans. The studies in this proposal are designed to identify the molecules which mediate haem homeostasis by measuring green fluorescent protein (GFP) fluorescence in live transgenic worms using a functional genome-wide reverse genetic RNAi screen in a haem sensor strain. The results from these studies will aid in delineating novel genes and molecules involved in eukaryotic haem homeostasis, and provide new insights into how iron and haem orchestrate developmental control in animals.
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Prof. Lois Smith
Children’s Hospital Boston, USA
The role of erythropoietin in retinopathy
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The blinding disease of proliferative retinopathy is characterised by an initial vessel loss phase followed by a hypoxia-induced destructive proliferation phase. The goal of this project is to determine the role of erythropoietin (EPO) in retinopathy and whether modulation of EPO can prevent retinopathy. EPO is an oxygen-regulated growth factor with a known function of stimulating erythrocyte production. EPO is widely administered for the treatment of anaemia in patients without regard to its effects on retinopathy. At present, the effect of EPO on retinal vascular stability is largely unknown. Using a mouse model of proliferative retinopathy, we found that EPO is important in retinal vascular growth. Early systemic supplementation of EPO during the vessel loss phase, when retinal EPO is deficient, significantly protected the retina from vessel degeneration, reducing the stimulus for subsequent proliferation. In contrast, late systemic supplementation of EPO during the second phase of retinopathy, when retinal EPO expression is already elevated, is not protective. These data suggest that the timing of EPO intervention is critical in the prevention of proliferative retinopathy. We propose to further investigate the role of EPO in the prevention of retinopathy by promoting normal vessel survival in the vessel loss phase of retinopathy. Using microarray analysis of laser captured vessels and neurons in EPO-treated and untreated retina, we will look for the factors whose expression in retina vessels (and neurons) are modulated by early EPO supplementation and determine their specific roles in vessel survival and proliferation. We will also investigate inhibition of pathological neovascularisation directly in the proliferative phase through inhibition of EPO with small interfering RNA (siRNA) gene silencing. Finally, we will examine if EPO is specifically regulated in diabetic mouse retinas and whether manipulation of EPO can prevent vessel loss in diabetic animals.
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