Role of iron for atherosclerosis

We hypothesize that tissue and especially macrophage iron overload due to alimentary (diet) and environmental (inflammation, chronic infection) factors promotes murine and human atherogenesis, and that these effects are modified by genetic polymorphisms predisposing to iron overload or influencing clearance of heme-derived iron from the vasculature. The epidemiological part will be conducted within the framework of the prospective population-based Bruneck Study in which development and progression of carotid atherosclerosis and manifestation of CVD have been carefully assessed over a 20-year period. We will analyze the relation of laboratory parameters reflecting extent and distribution of body iron with atherosclerosis and its clinical sequelae, the presence or absence of systemic inflammation, and frequent genetic mutations and polymorphisms affecting body iron homeostasis. Supplementary analyses will address vascular effects of iron depletion and modifiers of iron toxicity in the vasculature. For the experimental part, we will employ genetically modified mouse strains: Hfe-/- mice (model for hereditary hemochromatosis), and Apo-E deficient mice (model of lipid-inducible atherosclerosis) will be crossed and the resulting four groups will be challenged with diets containing varying amounts of iron to investigate the role of environmental and genetic iron overload (and their interaction) on atherogenesis. To study potential pro-atherogenic effects of macrophage iron overload induced by chronic inflammation additional Apo-E deficient mice will be injected intraperitoneally with LPS. In a comparable fashion we will cross Haptoglobin 2-2 (model for mice impaired iron retulization) and heme-oxygenase-1 (HO-1) knock out mice, respectively, with Apo-E deficient mice and expose them to the same challenges. Animal studies will focus on the extent of murine atherosclerosis and the expression of critical genes in atherogenesis, iron homeostasis and inflammation (within the lesions and systemically). The results of these combined human and animal studies will provide novel insights into the role of iron and inflammation in atherogenesis and have a high potential for clinical translation by applying readily available therapeutic measures to affect iron homeostasis upon iron chelation and phlebotomy.

Herein we analysed the association between disturbances of iron homeostasis (genetic and alimentary) along with environmental factors (inflammation, metabolic traits) on murine and human atherogenesis. The project consisted of two parts: The epidemiological part was conducted within the framework of the prospective population-based Bruneck Study in which development and progression of carotid atherosclerosis and manifestation of CVD has been carefully assessed over a 20-year period. We analysed the relation and causative association of laboratory parameters including hepcidin and lipocalin-2 with metabolic alterations (lipid and glucose metabolism) and extend of atherosclerosis and its clinical sequelae along with frequent genetic mutations affecting body iron homeostasis.The iron-containing molecule heme constituted the first focus of epidemiologic analyses. Heme, as part of haemoglobin, is essential for oxygen transport and contains the bulk of total body iron. However, heme circulating freely outside of red blood cells has well-known pro-oxidative, toxic properties, and may predispose to cardiovascular disease. We were able to show that subjects with certain well-defined variants in the gene encoding heme oxygenase-1, a key enzyme of heme degradation, face a high cardiovascular disease risk. These findings were novel and derived from the population-based Bruneck Study. In contrast, variants in haptoglobin, a circulating protein essential for removal of free haemoglobin from the circulation, did not associate with cardiovascular risk. Although prior studies reported that enrichment of haemoglobin by sugary moieties establishes or aggravates the association of haptoglobin with cardiovascular risk, our data was not consistent with any such effect modification.The second focus of our epidemiologic analyses revolved around hepcidin, the key hormonal regulator of systemic iron homeostasis. Although it has been reported in select patient groups that elevated hepcidin is capable of increasing cardiovascular disease risk, results from our random sample of the general community did not corroborate these findings. A further comprehensive characterisation of hepcidin in terms of its demographic and biochemical correlates found strong links to haemoglobin, liver function, inflammation, blood coagulation, alcohol consumption, and physical activity, and measured the relative importance of these factors. Finally, we showed that subjects with hepcidin levels that are inadequately low in relation to their total body iron content are at heightened risk of new-onset diabetes mellitus. This finding is of particular interest because diabetes is a major cardiovascular risk factor and hepcidin levels will be modifiable by drugs in the near future.For the supplementary experimental part we used atherosclerotic mice (ApoE) which were crossed with Hfe-/- mice, an establish model for genetic iron overload. We found that both, genetic and dietary iron challenge were assocaited with alterations of atherogenesis in the aorta along with subtle effects on lipid homeostasis. In addition, we were able to identify new modifiers of lipid homeostasis, so called lipoxin, which protect from the development of atherosclerotic lesions in mice. We also developed a second model by crossing ApoE with lipocalin-2 (Lcn2) -/- mice. Lcn2 is a small iron-siderophore binding peptide which exerts protection in ischemia- reperfusion injury. We found that Lcn2 stimulates the migration of neutrophils thereby triggering inflammatory reactions. Moreover, we found that Lcn2 plays decissive roles in the host responses towards the intracellualr bacterium C. pneumoniae, a pathogen which has been linked to atherogenesis. As macrophages are central for the development of atherosclerosis we studied pathways of iron homeostasis in these cells under inflamamtroy conditions. This lead to the identification of an nitric oxide controlled pathway by which macrophages export iron in response to the presence of intracellular bacteria. Our data generated within this project provided novel evidence for the assocation between atherhogenesis and underlying metabolic risk profils with genetic and environmental alterations of iron homeostasis. Our data elucidate novel risk factors for atherogenesis and encourage further studies aiming at modifing iron homeostasis to pervent or combat atherogenesis and its clinical sequels myocardial infarction and stroke.