Wieland, E. B.; Plug, A.; Balluff, B.; Gijbels, M.; Han, L.; Flinders, B.; Cuypers, E.; Kempen, L.; Li, X.; Mees, B.; Biessen, E. A.; Donners, M. M.; Goossens, P.

Intraplaque hemorrhage (IPH) is a hallmark of advanced atherosclerosis and a major risk factor for ischemic stroke and myocardial infarction. Current IPH classification focusses on extravascular erythrocyte presence as a proxy of acute bleeding, where iron detection generally indicates older hemorrhages. While often used interchangeably, a comprehensive analysis of transcriptional and metabolic context and impact of iron and erythrocyte deposition on the plaque is still lacking. Here, we investigate iron as a late-stage IPH hallmark in human atherosclerotic plaques. We analyzed erythrocyte-rich, iron-rich, and non-IPH regions in human carotid endarterectomy plaques by re-analyzing a published transcriptomic dataset of 43 patient samples. In addition, we performed histological and immune phenotyping to define plaque traits associated with iron versus erythrocyte accumulation. Finally, we performed spatial metabolic profiling to functionally define iron-rich regions. Although iron and erythrocyte deposits frequently co-localized, both co-related with different histological traits. While iron- and erythrocyte-rich regions shared transcriptomic features of advanced plaques compared with non-IPH regions, direct comparison showed differences in gene expression profiles. Iron deposition was associated with increased myeloid cell accumulation and a unique spatial metabolic signature distinct from erythrocyte-rich and non-IPH regions. While sharing many characteristics with IPH plaques, the molecular, cellular and metabolic landscape of iron-rich regions is marked by features of plaque remodeling and repair. This makes iron deposition a unique hallmark of late-stage IPH, extending the current erythrocyte-based definition of IPH.