MPI Phantom Study with A High-Performing Multicore Tracer Made by Coprecipitation

Magnetic particle imaging (MPI) is a new imaging technique that detects the spatial distribution of magnetic nanoparticles (MNP) with the option of high temporal resolution. MPI relies on particular MNP as tracers with tailored characteristics for improvement of sensitivity and image resolution. For this reason, we developed optimized multicore particles (MCP 3) made by coprecipitation via synthesis of green rust and subsequent oxidation to iron oxide cores consisting of a magnetite/maghemite mixed phase. MCP 3 shows high saturation magnetization close to that of bulk maghemite and provides excellent magnetic particle spectroscopy properties which are superior to Resovist® and any other up to now published MPI tracers made by coprecipitation. To evaluate the MPI characteristics of MCP 3 two kinds of tube phantoms were prepared and investigated to assess sensitivity, spatial resolution, artifact severity, and selectivity. Resovist® was used as standard of comparison. For image reconstruction, the regularization factor was optimized, and the resulting images were investigated in terms of quantifying of volumes and iron content. Our results demonstrate the superiority of MCP 3 over Resovist® for all investigated MPI characteristics and suggest that MCP 3 is promising for future experimental in vivo studies

Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT‐1 expression

Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. 'Uraemic' and control EV (EVUR; EVCTRL) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL, EC- and plasma-derived EVUR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22 alpha. Further, EVUR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EVUR. Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EVUR. In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs

Microdistribution of Magnetic Resonance Imaging Contrast Agents in Atherosclerotic Plaques Determined by LA-ICP-MS and SR-μXRF Imaging

Purpose: Contrast-enhanced magnetic resonance imaging (MRI) has the potential to replace angiographic evaluation of atherosclerosis. While studies have investigated contrast agent (CA) uptake in atherosclerotic plaques, exact CA spatial distribution on a microscale is elusive. The purpose of this study was to investigate the microdistribution of gadolinium (Gd)- and iron (Fe) oxide-based CA in atherosclerotic plaques of New Zealand White rabbits. Procedures: The study was performed as a post hoc analysis of archived tissue specimens obtained in a previous in vivo MRI study conducted to investigate signal changes induced by very small superparamagnetic iron oxide nanoparticles (VSOP) and Gd-BOPTA. For analytical discrimination from endogenous Fe, VSOP were doped with europium (Eu) resulting in Eu-VSOP. Formalin-fixed arterial specimens were cut into 5-μm serial sections and analyzed by immunohistochemistry (IHC: Movat’s pentachrome, von Kossa, and Alcian blue (pH 1.0) staining, anti-smooth muscle cell actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining) and elemental microscopy with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation μX-ray fluorescence (SR-μXRF) spectroscopy. Elemental distribution maps of Fe, Eu, Gd, sulfur (S), phosphorus (P), and calcium (Ca) were investigated. Results: IHC characterized atherosclerotic plaque pathomorphology. Elemental microscopy showed S distribution to match the anatomy of arterial vessel wall layers, while P distribution corresponded well with cellular areas. LA-ICP-MS revealed Gd and Fe with a limit of detection of ~ 0.1 nmol/g and ~ 100 nmol/g, respectively. Eu-positive signal identified VSOP presence in the vessel wall and allowed the comparison of Eu-VSOP and endogenous Fe distribution in tissue sections. Extracellular matrix material correlated with Eu signal intensity, Fe concentration, and maximum Gd concentration. Eu-VSOP were confined to endothelium in early lesions but accumulated in cellular areas in advanced plaques. Gd distribution was homogeneous in healthy arteries but inhomogeneous in early and advanced plaques. SR-μXRF scans at 0.5 μm resolution revealed Gd hotspots with increased P and Ca concentrations at the intimomedial interface, and a size distribution ranging from a few micrometers to submicrometers. Conclusions: Eu-VSOP and Gd have distinct spatial distributions in atherosclerotic plaques. While Eu-VSOP distribution is more cell-associated and might be used to monitor atherosclerotic plaque progression, Gd distribution indicates arterial calcification and might help in characterizing plaque vulnerability

Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study

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

Cardiac MR Elastography: Comparison with left ventricular pressure measurement

Purpose of the Study: To compare magnetic resonance elastography (MRE) with ventricular pressure changes in an animal model. Methods: Three pigs of different cardiac physiology (weight, 25 to 53 kg; heart rate, 61 to 93 bpm; left ventricular [LV] end-diastolic volume, 35 to 70 ml) were subjected to invasive LV pressure measurement by catheter and noninvasive cardiac MRE. Cardiac MRE was performed in a short-axis view of the heart and applying a 48.3-Hz shear-wave stimulus. Relative changes in LV-shear wave amplitudes during the cardiac cycle were analyzed. Correlation coefficients between wave amplitudes and LV pressure as well as between wave amplitudes and LV diameter were determined. Results: A relationship between MRE and LV pressure was observed in all three animals (R-square [greater than or equal to] 0.76). No correlation was observed between MRE and LV diameter (R-square [less than or equal to] 0.15). Instead, shear wave amplitudes decreased 102 +/- 58 ms earlier than LV diameters at systole and amplitudes increased 175 +/- 40 ms before LV dilatation at diastole. Amplitude ratios between diastole and systole ranged from 2.0 to 2.8, corresponding to LV pressure differences of 60 to 73 mmHg. Conclusion: Externally induced shear waves provide information reflecting intraventricular pressure changes which, if substantiated in further experiments, has potential to make cardiac MRE a unique noninvasive imaging modality for measuring pressure-volume function of the heart

Finite volume analysis of temperature effects induced by active MRI implants with cylindrical symmetry: 1. Properly working devices

BACKGROUND: Active Magnetic Resonance Imaging implants are constructed as resonators tuned to the Larmor frequency of a magnetic resonance system with a specific field strength. The resonating circuit may be embedded into or added to the normal metallic implant structure. The resonators build inductively coupled wireless transmit and receive coils and can amplify the signal, normally decreased by eddy currents, inside metallic structures without affecting the rest of the spin ensemble. During magnetic resonance imaging the resonators generate heat, which is additional to the usual one described by the specific absorption rate. This induces temperature increases of the tissue around the circuit paths and inside the lumen of an active implant and may negatively influence patient safety. METHODS: This investigation provides an overview of the supplementary power absorbed by active implants with a cylindrical geometry, corresponding to vessel implants such as stents, stent grafts or vena cava filters. The knowledge of the overall absorbed power is used in a finite volume analysis to estimate temperature maps around different implant structures inside homogeneous tissue under worst-case assumptions. The "worst-case scenario" assumes thermal heat conduction without blood perfusion inside the tissue around the implant and mostly without any cooling due to blood flow inside vessels. RESULTS: The additional power loss of a resonator is proportional to the volume and the quality factor, as well as the field strength of the MRI system and the specific absorption rate of the applied sequence. For properly working devices the finite volume analysis showed only tolerable heating during MRI investigations in most cases. Only resonators transforming a few hundred mW into heat may reach temperature increases over 5 K. This requires resonators with volumes of several ten cubic centimeters, short inductor circuit paths with only a few 10 cm and a quality factor above ten. Using MR sequences, for which the MRI system manufacturer declares the highest specific absorption rate of 4 W/kg, vascular implants with a realistic construction, size and quality factor do not show temperature increases over a critical value of 5 K. CONCLUSION: The results show dangerous heating for the assumed "worst-case scenario" only for constructions not acceptable for vascular implants. Realistic devices are safe with respect to temperature increases. However, this investigation discusses only properly working devices. Ruptures or partial ruptures of the wires carrying the electric current of the resonance circuits or other defects can set up a power source inside an extremely small volume. The temperature maps around such possible "hot spots" should be analyzed in an additional investigation

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles

Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI