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Oxidation-specific epitopes (OSE) within developing atherosclerotic lesions are key antigens that drive innate and adaptive immune responses in atherosclerosis, leading to chronic inflammation. Oxidized phospholipids and malondialdehyde-lysine epitopes are well-characterized OSE present in human atherosclerotic lesions, particularly in pathologically defined vulnerable plaques. Using murine and human OSE-specific antibodies as targeting agents, we have developed radionuclide and magnetic resonance based nanoparticles, containing gadolinium, manganese or lipid-coated ultrasmall superparamagnetic iron oxide, to non-invasively image OSE within experimental atherosclerotic lesions. These methods quantitate plaque burden, allow detection of lesion progression and regression, plaque stabilization, and accumulation of OSE within macrophage-rich areas of the artery wall, suggesting they detect the most active lesions. Future studies will focus on using "natural" antibodies, lipopeptides, and mimotopes for imaging applications. These approaches should enhance the clinical translation of this technique to image, monitor, evaluate efficacy of novel therapeutic agents, and guide optimal therapy of high-risk atherosclerotic lesions.

Purpose To determine if manganese (Mn) G8 dendrimers targeted to oxidation-specific epitopes (OSE) allow for in vivo detection of atherosclerotic lesions. Materials and Methods OSE have been identified as key factors in atherosclerotic plaque progression and destabilization. Mn offers a potentially clinically translatable alternative to gadolinium-based agents when bioretention and potential toxicity of gadolinium is anticipated. However, to be effective, high payloads of Mn must accumulate intracellularly in macrophages. It was hypothesized that G8 dendrimers targeted to OSE may allow delivery of high Mn payloads, thereby enabling in vivo detection of macrophage-rich plaques. G8 dendrimers were modified to allow conjugation with MnDTPA (758 Mn ion) and the antibody MDA2 that is targeted to malondialdehyde (MDA)-lysine epitopes. Both the untargeted and targeted G8 dendrimers were characterized and their in vivo efficacy evaluated in apoE−/− mice over a 96-hour time period after bolus administration of a 0.05 mmol Mn/kg dose using a clinical MR system (3T). Results Significant enhancement (normalized enhancement >60%, P = 0.0013) of atherosclerotic lesions was observed within a 72-hour time period following administration of the targeted dendrimers. The presence of Mn within atherosclerotic lesions was confirmed using spectroscopic methods (>8 μg Mn/g). Limited signal attenuation (

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

Objectives The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype. Background Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions. Methods Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively. Results In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p Conclusions These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

ObjectivesThis study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE).BackgroundOSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells.MethodsMultimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes (“targeted micelles”). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE−/−) and low-density lipoprotein receptor negative (LDLR−/−) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls.ResultsIn vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with ∼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE−/− and LDLR−/− mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles.ConclusionsThis study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.

Oxidative stress, and in particular oxidation of lipoproteins, is a hallmark of atherosclerosis. Upon entry of lipoproteins into the vessel wall, a cascade of pro-atherogenic pathways is initiated whereby the reaction of reactive oxygen species with substrates amenable to oxidation, such as polyunsaturated fatty acids, generates a variety of oxidation-specific epitopes on lipoproteins, proteins in the vessel wall, and apoptotic macrophages. Several of these oxidation-specific epitopes have been well characterized and specific murine and fully human antibodies have been generated in our laboratory to detect them in the vessel wall. We have developed radionuclide, gadolinium and iron oxide based MRI techniques to noninvasively image oxidation-specific epitopes in atherosclerotic lesions. These approaches quantitate plaque burden and also allow detection of atherosclerosis regression and plaque stabilization. In particular, gadolinium micelles or lipid-coated ultrasmall superparamagnetic iron oxide particles containing oxidation-specific antibodies accumulate within macrophages in the artery wall, suggesting they may image the most unstable plaques. Translation of these approaches to humans may allow a sensitive technique to image and monitor high-risk atherosclerotic lesions and may guide optimal therapeutic interventions.

Ex vivo generated dendritic cells are currently used to induce therapeutic immunity in solid tumors. Effective immune response requires dendritic cells to home and remain in lymphoid organs to allow for adequate interaction with T lymphocytes. The aim of the current study was to detect and track Feridex labeled human dendritic cells in murine models using magnetic resonance imaging. Human dendritic cells were incubated with Feridex and the effect of labeling on dendritic cells immune function was evaluated. Ex vivo dendritic cell phantoms were used to estimate sensitivity of the magnetic resonance methods and in vivo homing was evaluated after intravenous or subcutaneous injection. R2*-maps of liver, spleen, and draining lymph nodes were obtained and inductively coupled plasma mass spectrometry or relaxometry methods were used to quantify the Feridex tissue concentrations. Correlations between in vivo R2* values and iron content were then determined. Feridex labeling did not affect dendritic cell maturation or function. Phantom results indicated that it was possible to detect 125 dendritic cells within a given slice. Strong correlation between in vivo R2* values and iron deposition was observed. Importantly, Feridex-labeled dendritic cells were detected in the spleen for up to 2 weeks postintravenous injection. This study suggests that magnetic resonance imaging may be used to longitudinally track Feridex-labeled human dendritic cells for up to 2 weeks after injection.

Vulnerable or high-risk atherosclerotic plaques often exhibit large lipid cores and thin fibrous caps that can lead to deadly vascular events when they rupture. In this study, polyethylene glycol (PEG)-micelles that incorporate a gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) amphiphile were used as an MR contrast agent. In an approach inspired by lipoproteins, the micelles were functionalized with tyrosine residues, an aromatic, lipophilic amino acid, to reach the lipid-rich areas of atherosclerotic plaque in a highly efficient manner. These micelles were applied to apolipoprotein E(-/-) (ApoE(-/-)) mice as a model of atherosclerosis. The abdominal aortas of the animals were imaged using T(1)-weighted (T(1)W) high-resolution MRI at 9.4T before and up to 48 h after the administration of the micelles. PEG-micelles modified with 15% tyrosine residues yielded a significant enhancement of the abdominal aortic wall at 6 and 24 h postinjection (pi) as compared to unmodified micelles. Fluorescence microscopy on histological sections of the abdominal aorta showed a correlation between lipid-rich areas and the distribution of the functionalized contrast agent in plaque. Using a simple approach, we demonstrated that lipid-rich areas in atherosclerotic plaque of ApoE(-/-) mice can be detected by MRI using Gd-DTPA micelles.

Objectives The aim of the current study is to test the ability to label and detect murine embryonic stem cell–derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3). Background Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation. Methods ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3–labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy. Results In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed. Conclusions The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3–labeled ES-CPC in murine infarct models via CMR.

ObjectivesWe sought to determine whether gadolinium (Gd)-containing lipid-based nanoparticles (NPs) targeting the macrophage scavenger receptor-B (CD36) improve cardiac magnetic resonance (CMR) detection and characterization of human atherosclerosis.BackgroundGd-containing lipid-based NPs targeting macrophages have improved MR detection of murine atherosclerosis.MethodsGadolinium-containing untargeted NPs, anti-CD36 NPs, and nonspecific Fc-NPs were created. Macrophages were incubated with fluorescent targeted and nontargeted NPs to determine uptake via confocal microscopy and inductively coupled plasma mass spectroscopy (ICP-MS) quantified Gd uptake. Human aortic specimens were harvested at autopsy. With a 1.5-T scanner, T1, T2, and PDW 3-dimensional scans were performed along with post-contrast scans after 24 h incubation. The T1 and cluster analyses were performed and compared with immunohistopathology.ResultsThe NPs had a mean diameter of 125 nm and 14,900 Gd-ions, and relaxivity was 37 mmol/l−1s−1 at 1.5-T and 37°C. Confocal microscopy and ICP-MS demonstrated significant in vitro macrophage uptake of targeted NPs, whereas non-targeted NPs had minimal uptake. On T1 imaging, targeted NPs increased contrast-to-noise ratio (CNR) by 52.5%, which was significantly greater than Fc-NPs (CNR increased 17.2%) and nontargeted NPs (CNR increased 18.7%) (p = 0.001). Confocal fluorescent microscopy showed that NPs target resident macrophages, whereas the untargeted NPs and Fc-NPs are found diffusely throughout the plaque. Targeted NPs had a greater signal intensity increase in the fibrous cap compared with non-targeted NPs.ConclusionsMacrophage-specific (CD36) NPs bind human macrophages and improve CMR detection and characterization of human aortic atherosclerosis. Thus, macrophage-specific NPs could help identify high-risk human plaque before the development of an atherothrombotic event.

Determining arterial macrophage expression is an important goal in the molecular imaging of atherosclerosis. Here, we compare the efficacy of two synthetic, high density lipoprotein (HDL) based contrast agents for magnetic resonance imaging (MRI) of macrophage burden. Each form of HDL was labeled with gadolinium and rhodamine to allow MRI and fluorescence microscopy. Either the 37 or 18 amino acid peptide replaced the apolipoprotein A-I in these agents, which were termed 37pA-Gd or 18A-Gd. The diameters of 37pA-Gd and 18A-Gd are 7.6 and 8.0 nm, respectively, while the longitudinal relaxivities are 9.8 and 10.0 (mM s)(-1). 37pA has better lipid binding properties. In vitro tests with J774A.1 macrophages proved the particles possessed the functionality of HDL by eliciting cholesterol efflux and were taken up in a receptor-like fashion by the cells. Both agents produced enhancements in atherosclerotic plaques of apolipoprotein E knockout mice of approximately 90% (n = 7 per agent) and are macrophage specific as evidenced by confocal microscopy on aortic sections. The half-lives of 37pA-Gd and 18A-Gd are 2.6 and 2.1 h, respectively. Despite the more favorable lipid interactions of 37pA, both agents gave similar, excellent contrast for the detection of atherosclerotic macrophages using MRI

There is an ongoing desire to produce high-relaxivity, Gd-based magnetic resonance imaging (MRI) contrast agents. These may allow for lower doses to be used, which is especially important in view of the current safety concerns surrounding Gd in patients. Here we report the synthesis of a high-relaxivity MRI contrast agent, by incorporating Gd-chelating lipids that coordinate two water molecules into high-density lipoprotein (q = 2 HDL). We compared the properties of q = 2 HDL with those of an analogous HDL particle labeled with Gd-chelating lipids that coordinate only one water molecule (q = 1 HDL). We found that the q = 2 HDL possessed an elevated r(1) of 41 mM(-1) s(-1) compared to 9 mM(-1) s(-1) for q = 1 HDL at 20 MHz, but the q = 2 HDL exhibited high R(2)* values at high fields, precluding imaging above 128 MHz. While carrying out this investigation we observed that enlarged, disrupted particles were formed when the synthesis was carried out above the lipid critical micelle concentration (cmc), indicating the importance of synthesis below the cmc when modifying lipoproteins in this manner. The high relaxivity of q = 2 HDL means it will be an efficacious contrast agent for future MR imaging studies.

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.

In vivo markers that allow for detection of ferritin within atheromatous plaque may be useful for identifying iron-catalyzed hydroxyl-radical formation and subsequent lipid peroxidation. Recently, a positive contrast MR technique--GRadient echo Acquisition for Superparamagnetic particles/suscePtibility (GRASP)--was used to identify the presence of magnetic entities in phantom models. The aim of the current study was to determine the feasibility of using GRASP in conjunction with conventional T(2) (*)-weighted (T(2) (*)W) gradient-echo (GRE) sequences for identifying ferritin/hemosiderin deposition using in vitro and in vivo models of thrombus. In vitro thrombi were prepared by incubating blood with ferritin. MRI was performed using conventional GRE sequences and GRASP. The results indicate that GRASP was able to verify ferritin deposition in in vitro thrombi. In vivo thrombi were created using a crush injury model in rabbits. The signal enhancement obtained using conventional GRE sequences and GRASP was compared with the location of iron deposition by histology. In all of the animals the GRASP signal correlated with signal loss by conventional GRE, and ferritin/hemosiderin deposition by histology. GRASP sequences in combination with conventional GRE sequences may be used to detect the presence of ferritin deposition in in vitro thrombi and in vivo crush-injured rabbit carotid arteries.

Gadolinium (Gd) micelles are nanoparticles that incorporate phospholipids, surfactants, and lipophilic Gd complexes. Preliminary studies have shown that lipid-based nanoparticles may penetrate atherosclerotic plaque. The aim of the current study was to prepare, characterize, and evaluate in vivo the efficacy of two Gd micelle formulations using apolipoprotein E knockout (ApoE−/−) mouse models of atherosclerosis. Gd micelles were prepared using two different amphiphiles but similar GdDTPA lipids, surfactants, and fluorescent labels. The results indicate that the choice of amphiphile may affect the particle size, relaxivity, and blood clearance in wild-type mice (WT). However, the in vivo MR efficacy, with respect to uptake in the vessel wall of ApoE−/− mice, was not affected by the amphiphile used. Significant wall enhancement of ApoE−/− mice was observed following administration of 0.015 and 0.038 mmol Gd/kg of both micelle formulations. No significant enhancement of the vessel wall of WT mice was observed for any of the dosages or formulations tested. Additionally, liver uptake 24 hr post-injection (p.i.) was not influenced by the choice of amphiphile. The results of this study strongly suggest that liver uptake and wall enhancement may be regulated by the surface properties of the micelle and not by other factors, such as micelle size. Magn Reson Med, 2006. © 2006 Wiley-Liss, Inc.

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. T(2)* values were determined using multiple double-echo GRE. The GRASP sequence generated positive signal enhancement in phantoms containing iron. For all rephasing values

The oxidation of lipoproteins is a critical factor in the initiation, progression, and destabilization of atherosclerotic lesions. Reactive oxygen species and enzymes in the vessel wall modify lipoproteins to generate a variety of oxidation-specific epitopes (OSE). OSE are pro-inflammatory, immunogenic, and pro-atherogenic by activating macrophages leading to foam cell formation, apoptosis, and necrotic cores. OSE activate the innate and adaptive immune systems that lead to pro-inflammatory responses. The immunological properties of OSE have facilitated the generation and characterization and modification of murine and human monoclonal antibodies as imaging agents. Nuclear and magnetic resonance techniques have been used to visualize OSE in preclinical models. OSE-targeted antibodies have been derivatized with radioisotopes or complexed with gadolinium, manganese, and iron oxide nanoparticles to facilitate in vivo imaging of atherosclerotic lesions. OSE-targeted antibody uptake in the vessel wall is proportional to plaque burden in early and intermediate lesions allowing quantitation of the extent of atherosclerosis. In intervention studies, imaging of OSE allows the detection of atherosclerosis regression and features of plaque stabilization. Translation of these approaches to humans may provide sensitive techniques to image, diagnose, and monitor high-risk atherosclerotic lesions, guide optimal therapeutic interventions, and evaluate efficacy of novel therapeutic agents.

The identification and characterization of atherosclerotic lesions prone to rupture in asymptomatic individuals remains a major focus of diagnostic imaging research. Although ischemic events often result from severely stenotic lesions in the peripheral arteries, the majority of atherosclerotic

One of the several impediments to effective oncolytic virus therapy of cancer remains a lack of tumor-specific targeting. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells induced by tumor factors in tumor-bearing hosts. The biodistribution kinetics of MDSC and other immune cell types in a murine hepatic colon cancer model was investigated through the use of tracking markers and MRI. MDSCs were superior to other immune cell types in preferential migration to tumors in comparison with other tissues. On the basis of this observation, we engineered a strain of vesicular stomatitis virus (VSV), an oncolytic rhabdovirus that bound MDSCs and used them as a delivery vehicle. Improving VSV-binding efficiency to MDSCs extended the long-term survival of mice bearing metastatic colon tumors compared with systemic administration of wild-type VSV alone. Survival was further extended by multiple injections of the engineered virus without significant toxicity. Notably, direct tumor killing was accentuated by promoting MDSC differentiation towards the classically activated M1-like phenotype. Our results offer a preclinical proof-of-concept for using MDSCs to facilitate and enhance the tumor-killing activity of tumor-targeted oncolytic therapeutics. Cancer Res; 73(16); 5003–15. ©2013 AACR.

The purpose of this study was to test the hypothesis that increased oxidative stress is associated with apoptosis in human plaques with the haptoglobin (Hp) 2-2 genotype.Intraplaque hemorrhage releases free hemoglobin (Hb). Impaired Hb clearance induces oxidative stress leading to plaque progression. The binding of Hp to Hb attenuates iron-induced oxidative reactions.Twenty-six human aortic plaques were Hp genotyped. Hp2-2 plaques (n = 13) were compared with control (Hp1-1/2-1) (n = 13). The iron grade was measured by Perl's staining. Immunostaining was used to detect oxidation-specific epitopes (OSEs) reflecting oxidized phospholipids and malondialdehyde-like epitopes. The percentages of apoptotic cells and apoptotic morphological features were quantified. DNA fragmentation and active caspase-3 were measured by in situ end-labeling and immunohistochemistry, respectively.In Hp2-2 plaques, iron content was increased (1.22 ± 0.15 vs. 0.54 ± 0.08; p0.0001) along with expression of oxidized phospholipid- (78.9 ± 5.8 vs. 38.8 ± 3.8; p0.0001), and malondialdehyde-like OSEs (93.9 ± 7.9 vs. 54.7 ± 3.9; p0.0001). The total percentages of apoptotic cells (11.9 ± 0.44 vs. 3.5 ± 0.28; p0.0001), nuclear fragmentation (11.8 ± 0.50 vs. 3.3 ± 0.26; p0.0001), nuclear condensation (10.9 ± 0.58 vs. 3.4 ± 0.20; p0.0001), chromatin margination (14.2 ± 0.57 vs. 6.5 ± 0.37; p0.0001), cytoplasmic blebs (1.6 ± 0.28 vs. 0.8 ± 0.14; p0.002), and eosinophilia (10.8 ± 0.74 vs. 4.2 ± 0.27; p0.0001) were increased in Hp2-2 plaques. Furthermore, DNA fragmentation (119.9 ± 1.40 vs. 57.5 ± 0.80; p0.001), and active caspase-3 density (84.7 ± 7.62 vs. 50.6 ± 7.49; p0.004) were increased in Hp2-2 plaques. Logistic regression analysis identified correlation between the percentage of apoptotic cells and the density of OSEs (r = 0.56; p0.003).These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques.

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.

Ex vivo generated dendritic cells are currently used to induce therapeutic immunity in solid tumors. Effective immune response requires dendritic cells to home and remain in lymphoid organs to allow for adequate interaction with T lymphocytes. The aim of the current study was to detect and track Feridex labeled human dendritic cells in murine models using magnetic resonance imaging. Human dendritic cells were incubated with Feridex and the effect of labeling on dendritic cells immune function was evaluated. Ex vivo dendritic cell phantoms were used to estimate sensitivity of the magnetic resonance methods and in vivo homing was evaluated after intravenous or subcutaneous injection. R2*-maps of liver, spleen, and draining lymph nodes were obtained and inductively coupled plasma mass spectrometry or relaxometry methods were used to quantify the Feridex tissue concentrations. Correlations between in vivo R2* values and iron content were then determined. Feridex labeling did not affect dendritic cell maturation or function. Phantom results indicated that it was possible to detect 125 dendritic cells within a given slice. Strong correlation between in vivo R2* values and iron deposition was observed. Importantly, Feridex-labeled dendritic cells were detected in the spleen for up to 2 weeks postintravenous injection. This study suggests that magnetic resonance imaging may be used to longitudinally track Feridex-labeled human dendritic cells for up to 2 weeks after injection.

ObjectivesThe aim of this study was to determine whether iron oxide particles targeted to oxidation-specific epitopes image atherosclerotic lesions.BackgroundOxidized low-density lipoprotein plays a major role in atherosclerotic plaque progression and destabilization. Prior studies indicate that gadolinium micelles labeled with oxidation-specific antibodies allow for in vivo detection of vulnerable plaques with magnetic resonance imaging (MRI). However, issues related to biotransformation/retention of gadolinium might limit clinical translation. Iron oxides are recognized as safe and effective contrast agents for MRI. Because the efficacy of passively targeted iron particles remains variable, it was hypothesized that iron particles targeted to oxidation-specific epitopes might increase the utility of this platform.MethodsLipid-coated ultra-small superparamagnetic iron particles (LUSPIOs) (

Contrast-enhanced MRI of atherosclerosis can provide valuable additional information on a patient’s disease state. As a result of the interactions of HDL with atherosclerotic plaque and the flexibility of its reconstitution, it is a versatile candidate for the delivery of contrast-generating materials to this pathogenic lesion. We herein discuss the reports of HDL modified with gadolinium to act as an MRI contrast agent for atherosclerosis. Furthermore, HDL has been modified with fluorophores and nanocrystals, allowing it to act as a contrast agent for fluorescent imaging techniques and for computed tomography. Such modified HDL has been found to be macrophage specific, and, therefore, can provide macrophage density information via noninvasive MRI. As such, modified HDL is currently a valuable contrast agent for probing preclinical atherosclerosis. Future developments may allow the application of this particle to further diseases and pathological or physiological processes in both preclinical models as well as in patients.

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high-density lipoprotein (rHDL) nanoparticle platform enriched with Gd-based amphiphiles as a plaque-specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine-labeled apolipoprotein E-derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock-out (apoE−/−) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL–P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 µmol Gd/kg injection of rHDL–P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL–P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post-injection. Confocal laser scanning microscopy revealed that rHDL–P2A2 nanoparticles co-localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle. Copyright © 2008 John Wiley & Sons, Ltd.

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.

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.

Myocardial regeneration with stem-cell transplantation is a possible treatment option to reverse deleterious effects that occur after myocardial infarction. Since little is known about stem cell survival after transplantation, developing techniques for "tracking" cells would be desirable. Iron-oxide-labeled stem cells have been used for in vivo tracking using MRI but produce negative contrast images that are difficult to interpret. The aim of the current study was to test a positive contrast MR technique using reduced z-gradient rephasing (GRASP) to aid in dynamically tracking stem cells in an in vivo model of mouse myocardial infraction. Ferumoxides and protamine sulfate were complexed and used to magnetically label embryonic stem cell-derived cardiac-precursor-cells (ES-CPCs). A total of 500,000 ES-CPCs were injected in the border zone of infarcted mice and MR imaging was performed on a 9.4T scanner using T(2)*-GRE sequences (negative contrast) and positive contrast GRASP technique before, 24 hours, and 1 week after ES-CPC implantation. Following imaging, mice were sacrificed for histology and Perl's staining was used to confirm iron within myocardium. Good correlation was observed between signal loss seen on conventional T(2)* images, bright areas on GRASP, and the presence of iron on histology. This demonstrated the feasibility of in vivo stem cell imaging with positive contrast MRI.

International audience; PURPOSE: To test whether B-22956/1, a novel intravascular contrast agent with a high affinity to serum albumin (Bracco Imaging SpA.), allowed quantifying neovessel and macrophage density in atherosclerotic plaques of rabbits using MRI. MATERIALS AND METHODS: A T1-weighted MRI of the aorta was acquired in 10 rabbits (7 atherosclerotic and 3 control rabbits) before and up to 2 h after intravenous injection of 100 mumol/kg of Gd-DTPA or 75 mumol/kg of B-22956/1. Plaque enhancement was measured at different time points. Immunohistochemistry was performed using anti-CD 31 antibodies and anti-RAM 11 antibodies to correlate to neovessel and macrophage density, respectively. RESULTS: MRI showed a significant plaque enhancement 2 h after B-22956/1 versus Gd-DTPA in the atherosclerotic group (39.75% versus 9.5%; P < 0.0001. Early atherosclerotic plaques (n = 146) enhancement positively correlates with neovessel density on corresponding histological sections (r = 0.42; P < 0.01). Enhancement of atherosclerotic plaques 2 h after injection of B-22956/1 correlated with macrophage density (r = 0.71; P < 0.01). CONCLUSION: Enhancement of atherosclerotic plaques with MRI correlated with neovessel density at early time points after the injection of B-22956/1 and with macrophage density, at later time points. Hence, B-22956/1-enhanced MRI represents a promising imaging technique for the identification of "high-risk" plaques.

International audience; Macrophages have been identified as a critical factor in the pathogenesis of atherosclerosis. Ultrasmall iron oxide particles (USPIOs) have been used to passively target intraplaque macrophages. For dextran-based USPIOs, uptake into macrophages may be modulated by particle size. The aim of the current study was to test the efficacy of fractionated Feridex with respect to macrophage uptake in atherosclerotic rabbits. Fractionation of Feridex resulted in a 15-nm USPIO that exhibited a blood half-life of 15.9 h and liver retention of 6.4%. Blood clearance and liver retention of Feridex was 0.46 h and 60%, following administration of 4.8 mg Fe/kg Feridex. Atherosclerotic rabbits were administered 0.5 or 4.8 mg Fe/kg dosages of either fractionated Feridex or Feridex. MRI was performed at 1.5T over a 24-h time period postinjection. Perls and RAM-11 staining was performed to identify iron deposition. MRI showed a dose-dependent signal loss using conventional gradient echo (GRE) sequences following administration of fractionated Feridex. Even at low dose, significant signal loss was observed that correlated with histology. No signal attenuation or iron deposition was observed in the vessel wall of rabbits administered Feridex. Results of this study suggest that it may be possible to optimize USPIOs for intraplaque macrophage detection.

Background: Oxidized low-density lipoprotein (OxLDL) plays a key role in the initiation, progression and destabilization of atherosclerotic plaque. Molecular imaging probes that target OxLDL may allow in vivo detection of vulnerable plaques. In this study, magnetic resonance imaging (MRI) was used to detect atherosclerotic lesions in apolipoprotein deficient mice (ApoE−/−) using micelles comprised of gadolinium lipids, fluorescent rhodamine, PEG-lipids, and MDA2, a murine monoclonal antibody that binds malondialdehyde (MDA) lysine epitopes present in OxLDL. Materials and Results: Untargeted micelles, MDA2-labeled micelles and nonspecific polyclonal IgG micelles were prepared and characterized with respect to OxLDL binding capacity, pharmacokinetics, and biodistribution in wild type (WT) and ApoE−/− mice. MR imaging was performed at 9.4T over a 3-week interval after administration of 0.075 mmol Gd/kg micelles. MDA2 increased the micelle size, blood half-life, and MR efficacy relative to untargeted and IgG-micelles. Maximal plaque enhancement (>125%) was observed 72 hours post MDA2-micelle injection (Figure ). Untargeted and IgG-micelles did not exhibit significant wall enhancement at any of the time points studied. Confocal microscopy revealed that MDA2-micelles accumulate within foam cells associated with atherosclerotic plaque. WT mice showed no significant MR wall enhancement for any of the micelles studied. Conclusions: MR imaging using MDA2-micelles demonstrates specific targeting of OxLDL and foam cells and provides excellent MR image quality. This study suggests that it may be feasible to image similar atherosclerotic lesions in humans with MRI.

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.

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.

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 (78x39x78 microm3). The T1 value was significantly decreased in cells treated with micelles compared to Gd-DTPA (P

We sought to evaluate the effect of the particle size and coating material of various iron oxide preparations on the rate of rat liver clearance.The following iron oxide formulations were used in this study: dextran-coated ferumoxide (size = 97 nm) and ferumoxtran-10 (size = 21 nm), carboxydextran-coated SHU555A (size = 69 nm) and fractionated SHU555A (size = 12 nm), and oxidized-starch coated materials either unformulated NC100150 (size = 15 nm) or formulated NC100150 injection (size = 12 nm). All formulations were administered to 165 rats at 2 dose levels. Quantitative liver R2* values were obtained during a 63-day time period. The concentration of iron oxide particles in the liver was determined by relaxometry, and these values were used to calculate the particle half-lives in the liver.After the administration of a high dose of iron oxide, the half-life of iron oxide particles in rat liver was 8 days for dextran-coated materials, 10 days for carboxydextran materials, 14 days for unformulated oxidized-starch, and 29 days for formulated oxidized-starch.The results of the study indicate that materials with similar coating but different sizes exhibited similar rates of liver clearance. It was, therefore, concluded that the coating material significantly influences the rate of iron oxide clearance in rat liver.

Multicenter, randomized, placebo-controlled “outcome” trials with long-term follow-up of thousands of patients are currently being used to evaluate new therapies for cardiovascular disease. Owing to improvements in risk factor modifications as well as to the concomitant use of established treatments, such as statins, leading to a drop in clinical event rates, it is projected that the number of patients enrolled in trials may need to be increased to separate the effect of new therapies from those of established ones. Recently, the JUPITER (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) study (1) showed the importance of serologic measures of inflammation. These new in-blood markers may, however, be insufficient when used alone as predictive models of mortality and morbidity (2). Several recent cardiovascular trials have opted to use imaging (quantitative coronary angiography, carotid intima media thickness with ultrasonography, coronary intravascular ultrasonography, and so forth) to measure the impact of promising novel therapeutics (3). Validation of these in vivo diagnostic imaging methods may allow for shorter follow-up times and eventually, for smaller patient populations tested.

Vulnerable or high-risk atherosclerotic plaques often exhibit large lipid cores and thin fibrous caps that can lead to deadly vascular events via their rupture. In this study, PEGmicelles that incorporate a Gd-DTPA amphiphile were used as an MR contrast agent. In an approach inspired by lipoproteins, the micelles were functionalized with tyrosine residues, an aromatic, lipophilic amino acid, to target the lipid rich areas of atherosclerotic plaque in a highly efficient manner. Download : Download full-size image These micelles were applied to apolipoprotein E -/- mice as a model of atherosclerosis. The abdominal aortas of the animals were imaged using T1-weighted highresolution MRI at 9.4 T before and up to 48 hours after the administration of the micelles. PEG-micelles modified with 15 % tyrosine residues, yielded a significant enhancement of the abdominal aortic wall at 6 and 24 hours postinjection as compared to unmodified micelles. Fluorescence microscopy on histological sections of the abdominal aorta showed a correlation between lipid rich areas and the distribution of the functionalized contrast agent in plaque. Using a simple targeting approach, we demonstrated that lipid-rich areas in atherosclerotic plaque of ApoE -/- mice can be detected by MRI using Gd-DTPA micelles.