High Circulating Iron Levels Are a Risk Factor for Cardiovascular Disease: Clinical Implications for Iron-Overload Conditions
Authors: Vinchi Francesca , Simmelbauer Andreas , Costa da Silva Milene , Altamura Sandro , Galy Bruno , Hentze Matthias W. , Muckenthaler Martina U. 
Published: October 2019

Abstract
Pathologies associated with increased circulating iron levels include primary iron overload disorders, such as hereditary hemochromatosis, as well as secondary iron overload disorders, such as thalassemia, sickle cell anemia and myelodysplastic syndromes. In the latter elevated circulating iron levels due to enhanced intestinal iron absorption in support of ineffective erythropoiesis are further increased by the need of chronic blood transfusions. Organ toxicity and increased patient mortality under these conditions are mainly attributed to iron overload and the occurrence of non-transferrin-bound iron (NTBI). So far, epidemiological data and studies in animal models have provided conflicting evidence regarding a role of excess iron in atherogenesis and thrombosis. Here we investigate the role of high circulating iron levels in the development of cardiovascular disease.
A mouse model of type IV hereditary hemochromatosis, in which the hepcidin/ferroportin regulatory circuitry is disrupted due to a point mutation in the iron exporter ferroportin (Altamura et al., Cell Metabolism 2014), was interbred with ApoE-null mice, to study its susceptibility to atherosclerosis. As expected, this novel mouse model shows close to 100% transferrin saturation as well as NTBI. The mice were studied at 3, 6 and 12 months of age. We observed a mildly decreased lesion size and number at 3 months of age (6.15±1.25 vs 1.84±0.86 % aortic lesion area, P=0.02), while the same parameters were strongly increased in 6 to 12 month-old mice (6 months: 1.44±0.23 vs 5±0.53 % aortic lesion area, P=0.0001; 12 months: 10.24±1.21 vs 20.44±2.69 % aortic lesion area, P=0.0065). The atherosclerotic phenotype positively correlates with higher levels of circulating iron (12 months: 122.2±6.57 vs 337±19.22 mg iron/dl serum, P<0.0001) and oxidized LDLs (12 months: 2151±136.8 vs 3243±193.9 nmol oxLDL/ml serum, P=0.0002). In older mice the atherosclerotic plaques show reduced collagen deposition (P=0.0023) and increased lipid content (P=0.0495), indicating enhanced plaque instability and faster disease progression. Iron deposition in the artery media layer is associated with vascular smooth muscle cell senescence and alteration of the vascular oxidative status (12 months: 2.45±0.2 vs 4.33±0.32 nmol MDA/mg aorta protein, P=0.047). High circulating iron levels cause increased vascular permeability (6 months: 0.95±0.2 vs 3.11±0.51 mg aorta Evans Blue, P=0.0022), reduced nitric oxide availability and sustained activation and inflammation of the vascular endothelium (P<0.05). Our mouse model further shows increased levels of coagulation factors, such as fibrinogen and pro-thrombin (P<0.05), suggesting that high circulating iron levels may play a pro-thrombotic role. Interestingly, the number of plaque macrophages is significantly elevated due to increased iron-induced CCL2 levels (12 months: 155.5±23.27 vs 305±38.39 pmol CCL2/ml serum, P=0.01), potentially contributing to the enhanced vulnerability of the lesions (12 months, plaque vulnerability index: P=0.0276). It is of note that aortic macrophages are iron-depleted in this mouse model.
Our data suggest that in young mice (3 months) a less severe atherosclerotic phenotype may be explained by iron depletion of macrophages. With increasing age, high circulating iron levels strongly enhance the severity of the atherosclerotic phenotype, indicating that systemic iron overload is a risk factor for atherosclerosis progression and predisposes to cardiovascular disease.
Our findings have potential implications for those pathological conditions with elevated systemic iron levels, ranging from patients with hemochromatosis to anemic patients dependent on chronic blood transfusions, as well as for individuals subjected to intravenous iron administration (e.g. patients undergoing hemodialysis).

Disclosures
No relevant conflicts of interest to declare.