André M. N. Silva

AIMS: Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE-/- FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). METHODS AND RESULTS: Compared with normo-ferremic ApoE-/- mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE-/- FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE-/- FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. CONCLUSION: Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Citrate is an iron chelator and it has been shown to be the major iron ligand in the xylem sap of plants. Furthermore, citrate has been demonstrated to be an important ligand for the non-transferrin bound iron (NTBI) pool occurring in the plasma of individuals suffering from iron-overload. However, ferric citrate chemistry is complicated and a definitive description of its aqueous speciation at neutral pH remains elusive. X-Ray crystallography data indicates that the alcohol function of citrate (Cit4-) is involved in Fe(III) coordination and that deprotonation of this functional group occurs upon complex formation. The inability to include this deprotonation in the affinity constant calculations has been a major source of divergence between various reports of iron(III)-citrate affinity constants. However the recent determination of the alcoholic pKa of citric acid (H4Cit) renders the reassessment of the ferric citrate system possible. The aqueous speciation of ferric citrate has been investigated by mass spectrometry and EPR spectroscopy. It was observed that the most relevant species are a monoiron dicitrate species and dinuclear and trinuclear oligomeric complexes, the relative concentration of which depends on the solution pH value and the iron : citric acid molar ratio. Spectrophotometric titration was utilized for affinity constant determination and the formation constant for the biologically relevant [Fe(Cit)2]5- is reported for the first time.