Karen C. Briley-Saebo

Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI.

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium–diethyltriaminepentaacetic acid (DTPA) were tested in ApoE−/− and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE−/− mice compared with only 34% using untargeted micelles and no enhancement using gadolinium–DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture.

Gradient echo acquisition for superparamagnetic particles with positive contrast (GRASP): Sequence characterization in membrane and glass superparamagnetic iron oxide phantoms at 1.5T and 3T

Iron oxides are used for cell trafficking and identification of macrophages in plaque using MRI. Due to the negative contrast, differentiation between signal loss caused by iron and native low signal in tissue may be problematic. It is, therefore, preferable to achieve positive contrast. The purpose of this study was to test the efficacy of a new MRI sequence GRASP (GRe Acquisition for Superparamagnetic Particles) to generate a positive signal in phantoms containing iron. Membrane phantoms were constructed containing Ferumoxide at 7 concentrations. Standard GRE sequences were modified with user controlled z‐gradient rephasing (±100%). CNR values were determined as a function of echo time (TE) and % rephasing at 1.5T and 3T. T2* values were determined using multiple double‐echo GRE. The GRASP sequence generated positive signal enhancement in phantoms containing iron. For all rephasing values ≤30%, positive contrast was observed. The CNR generated at 1.5T was greater than the values at 3T for all concentrations tested. Correlation between CNR at 0% and 100% rephasing was observed at 1.5T(R = 0.84). Additionally, correlation between field change across the volume and CNR was observed. In conclusion, GRASP sequences may be used to generate positive signal enhancement in the presence of iron using MRI. Magn Reson Med, 2006.

Targeted Molecular Probes for Imaging Atherosclerotic Lesions With Magnetic Resonance Using Antibodies That Recognize Oxidation-Specific Epitopes

Background— Oxidized low-density lipoprotein plays a key role in the initiation, progression, and destabilization of atherosclerotic plaques and is present in macrophages and the lipid pool. The aim of this study was to assess the feasibility of magnetic resonance imaging of atherosclerotic lesions in mice using micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bind unique oxidation-specific epitopes. Methods and Results— MDA2 micelles, E06 micelles, IK17 micelles, nonspecific IgG micelles, and untargeted micelles (no antibody) were prepared and characterized with respect to pharmacokinetics and biodistribution in wild-type and atherosclerotic apolipoprotein E–deficient (apoE−/−) mice. Magnetic resonance imaging was performed at 9.4 T over a 96-hour time interval after the administration of 0.075–mmol Gd/kg micelles. MDA2, E06, and IK17 micelles exhibited a longer plasma half-life than IgG or untargeted micelles in apoE−/− but not wild-type mice. In apoE−/− mice, MDA2 and IK17 micelles showed maximal arterial wall uptake at 72 hours and E06 micelles at 96 hours, manifested by 125% to 231% enhancement in magnetic resonance signal compared with adjacent muscle. Confocal microscopy revealed that MDA2, IK17, and E06 micelles accumulated within atherosclerotic lesions and specifically within macrophages. Intravenous injection of free MDA2 before imaging with MDA2 micelles resulted in significantly diminished magnetic resonance signal enhancement. IgG micelles and untargeted micelles showed minimal enhancement in apoE−/− mice. There was no significant signal enhancement with all micelles in wild-type mice. Conclusions— Magnetic resonance imaging with micelles containing gadolinium and oxidation-specific antibodies demonstrates specific targeting and excellent image quality of oxidation-rich atherosclerotic lesions.

MRI to detect atherosclerosis with gadolinium-containing immunomicelles targeting the macrophage scavenger receptor.

The ability to specifically image macrophages may enable improved detection and characterization of atherosclerosis. In this study we evaluated the in vitro uptake of gadolinium (Gd)‐containing immunomicelles (micelles linked to macrophage‐specific antibody), micelles, and standard contrast agents by murine macrophages, and sought to determine whether immunomicelles and micelles improve ex vivo imaging of apolipoprotein E knockout (ApoE KO) murine atherosclerosis. Murine RAW 264.7 macrophages were incubated with Gd‐DTPA, micelles, and immunomicelles. Cell pellets were prepared and imaged using a 1.5 T MR system with an inversion recovery spin‐echo sequence to determine the in vitro T1 values. Ex vivo analysis of mouse aortas was performed using a 9.4T MR system with a high‐spatial‐resolution sequence (78 × 39 × 78 μm3). The T1 value was significantly decreased in cells treated with micelles compared to Gd‐DTPA (P < 0.0001), and in cells incubated at 4°C with immunomicelles compared to micelles (P < 0.05). Ex vivo MRI signal intensity (SI) was significantly increased by 81% and 20% in aortas incubated with immunomicelles and micelles, respectively. Confocal microscopy demonstrated in vitro and ex vivo uptake of fluorescent immunomicelles by macrophages. Immunomicelles and micelles improve in vitro and ex vivo MR detection of macrophages, and may prove useful in the detection of macrophage‐rich plaques. Magn Reson Med, 2006.

Magnetic Resonance Imaging of Vulnerable Atherosclerotic Plaques: Current Imaging Strategies and Molecular Imaging Probes

The vulnerability or destabilization of atherosclerotic plaques has been directly linked to plaque composition. Imaging modalities, such as magnetic resonance (MR) imaging, that allow for evaluation of plaque composition at a cellular and molecular level, could further improve the detection of vulnerable plaque and may allow for monitoring the efficacy of antiatherosclerotic therapies. In this review we focus on MR imaging strategies for the detection and evaluation of atherosclerotic plaques and their composition. We highlight recent advancements in the development of MR pulse sequences, computer image analysis, and the use of commercially available MR contrast agents, such as gadopentic acid (Gd‐DTPA), for plaque characterization. We also discuss molecular imaging strategies that are currently being used to design specific imaging probes targeted to biochemical and cellular markers of atherosclerotic plaque vulnerability. J. Magn. Reson. Imaging 2007;26:460–479.

Detection of Neovessels in Atherosclerotic Plaques of Rabbits Using Dynamic Contrast Enhanced MRI and 18F-FDG PET

Objective—The association of inflammatory cells and neovessels in atherosclerosis is considered a histological hallmark of high-risk active lesions. Therefore, the development and validation of noninvasive imaging techniques that allow for the detection of inflammation and neoangiogenesis in atherosclerosis would be of major clinical interest. Our aim was to test 2 techniques, black blood dynamic contrast enhanced MRI (DCE-MRI) and 18-fluorine-fluorodeoxyglucose (18F-FDG) PET, to quantify inflammation expressed as plaque neovessels content in a rabbit model of atherosclerosis. Methods and Results—Atherosclerotic plaques were induced in the aorta of 10 rabbits by a combination of 2 endothelial abrasions and 4 months hyperlipidemic diet. Six rabbits underwent MRI during the injection of Gd-DTPA, whereas 4 rabbits were imaged after injection of 18F-FDG with PET. We found a positive correlation between neovessels count in atherosclerotic plaques and (1) Gd-DTPA uptake parameters evaluated by DCE-MRI (r=0.89, P=0.016) and (2) 18F-FDG uptake evaluated by PET (r=0.5, P=0.103 after clustered robust, Huber-White, standard errors analysis). Conclusion—DCE-MRI and 18F-FDG PET may allow for the evaluation of inflammation in atherosclerotic plaques of rabbits. These noninvasive imaging modalities could be proposed as clinical tools in the evaluation of lesion prognosis and monitoring of anti–angiogenic therapies.

NC100150 injection, a preparation of optimized iron oxide nanoparticles for positive-contrast MR angiography

A preparation of monocrystalline iron oxide nanoparticles with an oxidized starch coating, currently in clinical trials (NC100150 Injection; CLARISCAN™), was characterized by magnetization measurements, relaxometry, and photon correlation spectroscopy. By combining the results with a measure of iron content, one can obtain the size and magnetic attributes of the iron cores, including the relevant correlation times for outer sphere relaxation (τSO and τD), and information about the interaction of the organic coating with both core and solvent. The results are 6.43 nm for the iron oxide core diameter, a magnetic moment of 4.38 × 10−17 erg/G, and a water‐penetrable coating region of oxidized oligomeric starch fragments and entrained water molecules. The latter extends the hydrodynamic diameter to 11.9 nm and lowers the average diffusivity of solvent about 64% (which increases τD accordingly). The nanoparticles show little size‐polydispersity, evidenced by the lowest value of r2/r1 at 20 MHz reported to date, an asset for magnetic resonance angiography. J. Magn. Reson. Imaging 2000;11:488–494.

Crystal size and properties of superparamagnetic iron oxide (SPIO) particles

The properties of a superparamagnetic iron oxide (SPIO) model contrast agent have been studied. The test material, HEP-SPIO, contained iron oxide multicrystal agglomerates coated with heparin, polyanionic, naturally occurring glycosaminoglycan. Fractionation of the HEP-SPIO suspension showed the existence of colloidally stable particles ranging from approx. 100 nm down to single crystal sizes. The small (< 20 nm) particles represented the major number fraction of particles present, but only approx. 2% of the total iron oxide mass. The volume weighted average diameter of the individual iron oxide crystals forming the multicrystal agglomerates was found to be 11-12 nm using transmission electron microscopy and vibrating sample magnetometry (VSM) techniques. Comparable results were obtained with X-ray diffraction and Mössbauer spectroscopy. A number of additional SPIO properties could also be determined on a routine VSM, such as the distribution standard deviation for the log-normal distribution of crystal sizes, the magnetic susceptibility, the magnetic remanence, and the intrinsic magnetization (magnetic moment) of the iron oxide. These parameters are useful tools for evaluation of the magnetic characteristics and contrast efficacy of SPIO contrast agents.

Assessment of T1 and T2* effects in vivo and ex vivo using iron oxide nanoparticles in steady state : dependence on blood volume and water exchange

Accurate knowledge of the relationship between contrast agent concentration and tissue relaxation is a critical requirement for quantitative assessment of tissue perfusion using contrast‐enhanced MRI. In the present study, using a pig model, the relationship between steady‐state blood concentration levels of an iron oxide nanoparticle with a hydrated diameter of 12 nm (NC100150 Injection) and changes in the transverse and longitudinal relaxation rates (1/T  *2 and 1/T1, respectively) in blood, muscle, and renal cortex was investigated at 1.5 T. Ex vivo measurements of 1/T  *2 and 1/T1 were additionally performed in whole pig blood spiked with different concentrations of the iron oxide nanoparticle. In renal cortex and muscle, 1/T  *2 increased linearly with contrast agent concentration with slopes of 101 ± 22 s−1mM−1 and 6.5 ± 0.9 s−1mM−1 (mean ± SD), respectively. In blood, 1/T  *2 increased as a quadratic function of contrast agent concentration, with different quadratic terms in the ex vivo vs. the in vivo experiments. In vivo, 1/T1 in blood increased linearly with contrast agent concentration, with a slope (T1‐relaxivity) of 13.9 ± 0.9 s−1mM−1. The achievable increase in 1/T1 in renal cortex and muscle was limited by the rate of water exchange between the intra‐ and extravascular compartments and the 1/T1‐curves were well described by a two‐compartment water exchange limited relaxation model. Magn Reson Med 47:461–471, 2002.

An ApoA-I mimetic peptide high-density-lipoprotein-based MRI contrast agent for atherosclerotic plaque composition detection

Cardiovascular disease is one of the prime causes of mortality throughout the world and there is a need for targeted and effective contrast agents to allow noninvasive imaging of the cholesterol-rich atherosclerotic plaques in arteries. A new, fully synthetic, high-density lipoprotein (HDL)-mimicking MRI contrast agent is developed, which enhances macrophage-rich areas of plaque in a mouse model of atherosclerosis by 94%. Confirmation of the targeting of this nanoparticulate agent is achieved using confocal microscopy by tracking a fluorescent lipid incorporated into the nanoparticle.