The study investigates the role of cell-surface glycosaminoglycans during the rapid uptake of Synomag®-COOH, a promising MPI-tracer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Introduction: Atherosclerosis is the underlying cause of multiple cardiovascular pathologies. The present-day clinical imaging modalities do not offer sufficient information on plaque composition or rupture risk. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a strongly upregulated proteoglycan-cleaving enzyme that is specific to cardiovascular diseases, inter alia, atherosclerosis. Materials and Methods: Male apolipoprotein E-deficient mice received a high-fat diet for 2 (n = 11) or 4 months (n = 11). Additionally, a group (n = 11) receiving pravastatin by drinking water for 4 months alongside the high-fat diet was examined. The control group (n = 10) consisted of C57BL/6J mice on standard chow. Molecular magnetic resonance imaging was performed prior to and after administration of the gadolinium (Gd)-based ADAMTS4-specific probe, followed by ex vivo analyses of the aortic arch, brachiocephalic arteries, and carotid arteries. A P value <0.05 was considered to indicate a statistically significant difference. Results: With advancing atherosclerosis, a significant increase in the contrast-to-noise ratio was measured after intravenous application of the probe (mean precontrast = 2.25; mean postcontrast = 11.47, P < 0.001 in the 4-month group). The pravastatin group presented decreased ADAMTS4 expression. A strong correlation between ADAMTS4 content measured via immunofluorescence staining and an increase in the contrast-to-noise ratio was detected (R ² = 0.69). Microdissection analysis revealed that ADAMTS4 gene expression in the plaque area was significantly greater than that in the arterial wall of a control mouse (P < 0.001). Laser ablation–inductively coupled plasma–mass spectrometry confirmed strong colocalization of areas positive for ADAMTS4 and Gd. Conclusions: Magnetic resonance imaging using an ADAMTS4-specific agent is a promising method for characterizing atherosclerotic plaques and could improve plaque assessment in the diagnosis and treatment of atherosclerosis.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Inactive rhomboid protein 2 (iRhom2) regulates ADAM17-mediated shedding of tumor necrosis factor-α (TNF-α) from immune cells. We previously showed that iRhom2 deficiency attenuates early atherosclerosis in mice. This study aimed to characterize the impact of iRhom2 deficiency on macrophage phenotype and function. Bone marrow-derived macrophages (BMDMs) from iRhom2^−/− and iRhom2^+/+ mice were analyzed for proliferation, phagocytosis, survival of cytotoxic stress, and polarization. Cytokine secretion after LPS stimulation was quantified, and iRhom2 expression under atherogenic stimuli was assessed. Conditioned media from BMDMs (BMDMcM) were applied to human aortic endothelial cells (HAoECs) to evaluate adhesion molecule expression and monocyte adhesion. iRhom2 deficiency did not affect BMDM proliferation, phagocytosis, survival, or polarization marker expression. iRhom2 expression was upregulated in iRhom2^+/+ BMDMs by atherogenic stimulation. Following LPS stimulation, TNF-α secretion was decreased and IL-10 secretion was increased in iRhom2^−/− compared with iRhom2^+/+ BMDMs. HAoEC expression of adhesion molecules—ICAM-1, VCAM-1, and E-selectin—was attenuated after exposure to iRhom2^−/− compared with iRhom2^+/+ BMDMcM. Monocyte adhesion to HAoECs was reduced following treatment with iRhom2^−/− BMDMcM; TNF-α neutralization abolished this effect, indicating TNF-α dependency. iRhom2 deficiency in BMDMs selectively alters macrophage inflammatory cytokine secretion without affecting basal macrophage functions, thereby reducing endothelial activation and monocyte adhesion. These findings identify iRhom2 as a regulator of macrophage–endothelial crosstalk and a potential target to modulate inflammation in atherogenesis.