Cell response to imaging contrast agents suggested for atherosclerotic plaque imaging

Abstract: Oxysterols are the major cytotoxic components of oxidized low-density lipoprotein (OxLDL) that accumulate in atherosclerotic plaques. Their uptake by macrophages ensue foam cell formation, atherogenesis and plaque progression. Magnetic resonance imaging (MRI) has grown as a modality to track such intra-plaque developments by using intracellular contrast agents. The focus of this study was to evaluate the effects of two contrast agents; manganese based mangafodipir (TeslascanTM) and iron based super-paramagnetic iron oxide nanoparticles (SPION, ResovistTM) on cell functions and examined their interaction with oxysterol laden cells.Mangafodipir has antioxidant property and provides protection against oxidative stress. The chemical structure of mangafodipir comprises of organic ligand fodipir (Dipyridoxyl diphosphate, Dp-dp) and Mn (manganese). Mangafodipir is readily metabolized within the body to manganese dipyridoxyl ethyldiamine (MnPLED) after an intravenous injection. MnPLED has superoxide dismutase (SOD) mimetic activity, and Dp-dp has iron chelating effects. The second contrast agent tested in this study is ResovistTM. These SPION are primarily ingested by macrophages and accumulated in lysosomes where they are gradually degraded ensuing increased cellular iron.In paper I, we examined whether the above-noted effects of mangafodipir could be utilized to prevent 7β-hydroxycholesterol (7βOH) induced cell death. We found that mangafodipir prevents 7βOH induced cell death by attenuating reactive oxygen species (ROS) and by preserving lysosomal membrane integrity and mitochondrial membrane potential.The second part of this study (paper II) was designed to identify the pharmacologically active part of mangafodipir, which exerts the above-noted effects. We compared the activity of parent compound (mangafodipir) with MnPLED and Dp-dp. We found that mangafodipir; MnPLED and Dp-dp provide similar cyto-protection against 7βOH induced cell death. These results suggest that MnPLED and Dp-dp both contribute to the pharmacologically active part of mangafodipir.In paper III, we aimed to examine the interaction of SPION with monocytes and macrophages exposed or not to atheroma relevant oxysterols. We demonstrate that SPION loading up-regulates cellular levels of cathepsin and ferritin and induces membranous ferroportin expression. Additionally, SPION incites secretion of ferritin and both pro-inflammatory and anti-inflammatory cytokines. Moreover, exposure to oxysterols resulted in a reduced SPION uptake by cells, which may lead to inefficient targeting of such cells. Although SPION uptake was reduced, the ingested amounts significantly up-regulated the expression of 7βOH induced cathepsin B, cathepsin L and ferritin in cells, which may further aggravate atherogenesis.The fourth part of the study (paper IV) was designed to examine the interaction of SPION with macrophage subtypes and compare the cellular effects of coated and uncoated iron-oxide nanoparticles. We found that iron in SPION induces a phenotypic shift in THP1 M2 macrophages towards a macrophage subtype characterized by upregulated intracellular levels of CD86, ferritin and cathepsin L. Differential levels of these proteins among macrophage subtypes might be important to sustain a functional plasticity. Additionally, uncoated iron-oxide nanoparticles induced dose dependent cell death in macrophages, which elucidates the potential cyto-toxicity of iron in iron-oxide nanoparticles.In conclusion, evidence is provided in this study that intracellular MRI contrast agents have the potential to modulate cell functions. The study reveals a therapeutic potential of mangafodipir, which could be utilized for future development of contrast agents with both diagnostic and curative potentials. Additionally, we found that surface coating in SPION may provide cell tolerance to iron toxicity by modulation of cellular iron metabolism and cell functions. Such alterations in cellular metabolism call for careful monitoring and also highlight new concepts for development of iron containing nanoparticles. A reduced uptake of SPION by atheroma relevant cells justifies development of functionalized SPION to target such cells in atherosclerotic plaques.