Aims (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidemic models of atherosclerosis. Methods and results RAW264.7 foam cells, exposed in vitro to Ferumoxide (dextran-coated SPIO), Ferumoxtran (dextran-coated USPIO), or Ferumoxytol (carboxymethyl dextran-coated USPIO) (all 1 mg Fe/ml) showed increased apoptosis and ROS accumulation for Ferumoxide and Ferumoxtran, whereas Ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of Ferumoxide (0.3 mg Fe/kg) and Ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9–16/group) mice that had received single IV injections compared to saline-treated controls. Again, Ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (−68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P  < 0.001, n = 8/group). Repeated Ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received Ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed 5-fold increased apoptosis (18.2 vs. 3.7% respectively; P = 0.004) compared to controls who did not receive Ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of Ferumoxide and Ferumoxtran. Conclusions Ferumoxide and Ferumoxtran, but not Ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.

Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1β, IL-8, and TNF-α. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 103 cells/mm2), as visualized by principal component analysis. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments.

Monocytes circulate the vasculature at steady state and are recruited to sites of inflammation where they differentiate into macrophages (MФ) to replenish tissue-resident MФ populations and engage in the development of cardiovascular disease (CVD). Monocytes display considerable heterogeneity, currently reflected by a nomenclature based on their expression of cluster of differentiation (CD) 14 and CD16, distinguishing CD14++CD16- classical (cMo), CD14++CD16+ intermediate (intMo) and CD14 + CD16++ non-classical (ncMo) monocytes. Several reports point to shifted subset distributions in the context of CVD, with significant association of intMo numbers with atherosclerosis, myocardial infarction, and heart failure. However, clear indications of their causal involvement as well as their predictive value for CVD are lacking. As recent high-parameter cytometry and single-cell RNA sequencing (scRNA-Seq) studies suggest an even higher degree of heterogeneity, better understanding of the functionalities of these subsets is pivotal. Considering their high heterogeneity, surprisingly little is known about functional differences between MФ originating from monocytes belonging to different subsets, and implications thereof for CVD pathogenesis. This paper provides an overview of recent findings on monocyte heterogeneity in the context of homeostasis and disease as well as functional differences between the subsets and their potential to differentiate into MФ, focusing on their role in vessels and the heart. The emerging paradigm of monocyte heterogeneity transcending the current tripartite subset division argues for an updated nomenclature and functional studies to substantiate marker-based subdivision and to clarify subset-specific implications for CVD.