Magnetic resonance imaging contrast agent targeted toward activated platelets allows in vivo detection of thrombosis and monitoring of thrombolysis.

BACKGROUND: Platelets are the key to thrombus formation and play a role in the development of atherosclerosis. Noninvasive imaging of activated platelets would be of great clinical interest. Here, we evaluate the ability of a magnetic resonance imaging (MRI) contrast agent consisting of microparticles of iron oxide (MPIOs) and a single-chain antibody targeting ligand-induced binding sites (LIBS) on activated glycoprotein IIb/IIIa to image carotid artery thrombi and atherosclerotic plaques. METHODS AND RESULTS: Anti-LIBS antibody or control antibody was conjugated to 1-microm MPIOs (LIBS MPIO/control MPIO). Nonocclusive mural thrombi were induced in mice with 6% ferric chloride. MRI (at 9.4 T) was performed once before and repeatedly in 12-minute-long sequences after LIBS MPIO/control MPIO injection. After 36 minutes, a significant signal void, corresponding to MPIO accumulation, was observed with LIBS MPIOs but not control MPIOs (P<0.05). After thrombolysis, in LIBS MPIO-injected mice, the signal void subsided, indicating successful thrombolysis. On histology, the MPIO content of the thrombus, as well as thrombus size, correlated significantly with LIBS MPIO-induced signal void (both P<0.01). After ex vivo incubation of symptomatic human carotid plaques, MRI and histology confirmed binding to areas of platelet adhesion/aggregation for LIBS MPIOs but not for control MPIOs. CONCLUSIONS: LIBS MPIOs allow in vivo MRI of activated platelets with excellent contrast properties and monitoring of thrombolytic therapy. Furthermore, activated platelets were detected on the surface of symptomatic human carotid plaques by ex vivo MRI. This approach represents a novel noninvasive technique allowing the detection and quantification of platelet-containing thrombi.

Superparamagnetic iron oxide binding and uptake as imaged by magnetic resonance is mediated by the integrin receptor Mac-1 (CD11b/CD18): implications on imaging of atherosclerotic plaques.

INTRODUCTION: Superparamagnetic iron oxide nanoparticles (SPIONs) have been successfully used for magnetic resonance imaging (MRI) of atherosclerotic plaques. Endocytosis into monocytes/macrophages has been proposed as the mechanism for SPION uptake, but a specific receptor has not been identified yet. A potential candidate is the versatile integrin Mac-1 (CD11b/CD18, alphaMbeta2), which is involved in leukocyte adhesion, complement activation and phagocytosis. METHODS AND RESULTS: Intracellular SPION-accumulation was confirmed in cultured human monocytes using immunohistochemistry and iron staining. Recombinant cells expressing Mac-1 in different activation states as well as human monocytes with or without PMA stimulation were incubated either with an unspecific IgG or a CD11b-blocking antibody. Thereafter, cells were incubated with FITC-labeled amino-covered SPIONs or ferumoxtran-10 SPIONs and signal intensity was quantified by flow cytometry. Depending on the activation status of Mac-1, a significant increase in SPION binding/uptake was observed, independent on surface coating. Furthermore, SPION binding/uptake was significantly reduced after CD11b blockade. Results were confirmed in recombinant cells incubated with amino-PVA SPIONs and ferumoxtran-10, using T2(*)-weighted 3T MRI. CONCLUSION: The integrin Mac-1 is directly involved in SPION binding/uptake. Thus, monocytes abundantly expressing Mac-1 and especially activated monocytes expressing activated Mac-1 may be useful vehicles for high resolution MRI labeling of atherosclerotic plaques.

Imaging monocytes with iron oxide nanoparticles targeted towards the monocyte integrin MAC-1 (CD11b/CD18) does not result in improved atherosclerotic plaque detection by in vivo MRI.

Imaging of macrophages with superparamagnetic iron oxide particles (SPIO) has been performed to improve detection of atherosclerotic plaque inflammation in human and mouse studies by molecular magnetic resonance imaging (MRI). Since affinity of the monocyte/macrophage integrin MAC-1 (CD11b/CD18) is upregulated in inflammation, we generated a contrast agent targeting CD11b (CD11b-SPIOs) for improved macrophage detection in plaques. CD11b-SPIOs and non-targeted SPIOs (control-SPIOs) were incubated in vitro with human monocytes/macrophages. As quantified by SPIO-induced MRI signal extinction, intracellular iron-content was significantly higher in monoytes/macrophages incubated with CD11b-SPIO than with control-SPIO in vitro (p < 0.05), suggesting an improved uptake of CD11b-SPIOs into monocytes. Therefore, the aortic arch (AA) and vessel branches of ApoE(-/-)-knockout mice on a Western-type diet were imaged before and 48 h after contrast agent injection of either CD11b-SPIOs or control-SPIOs, using a 9.4 T animal MRI system. The SPIO-induced change in the MRI signal was quantified, as well as the macrophage-content by anti-CD68 immunhistochemistry and the iron-content by Prussian-blue staining. However, SPIO-induced signal extinction in in vivo-MRI was similar in CD11b-SPIO and control-SPIO-injected animals, with a non-significant trend towards an improved uptake of CD11b-SPIOs in the subclavian artery and subsections of the AA. These data correlated well with the results obtained by histology. Although in vitro MRI-data indicated an increased uptake of targeted CD11b-SPIOs in monocytes/macrophages, in vivo mouse data do not allow improved atherosclerotic plaque detection compared WITH non-targeted SPIOs. Therefore, CD11b-targeted MRI contrast labelling of monocytes/macrophages does not seem to be a successful strategy in stable atherosclerotic plaques such as found in the ApoE(-/-)-knockout-model. However, the impressive correlation between MRI and histology data encourages further development of inflammation- and plaque-specific contrast agents for vulnerable plaque imaging.

Purification and amino acid sequence of aminopeptidase P from pig kidney.

Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized.