In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Biocompatible Manganese Molecular Magnetic Imaging Probes

Abstract

Objectives To evaluate the in vivo magnetic resonance (MR) imaging efficacy of manganese (Mn(II)) molecular imaging probes targeted to oxidation-specific epitopes (OSE). Background OSE are critical in the initiation, progression and de-stabilization of atherosclerotic plaques. Gadolinium (Gd(III)) based MR imaging agents can be associated with systemic toxicity. Mn is an endogenous, bio-compatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells. Methods Multimodal 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 Tesla) evaluated in apoE−/− and 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. Results In vitro studies demonstrated that targeted Mn-micelles accumulate in macrophages when pre-exposed to MDA-LDL with ~10X increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48–72 hours after administration of targeted Mn-micelles, with co-localization 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. Conclusions This study demonstrates that bio-compatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of non-invasive imaging of human atherosclerosis.

Differential expression of oxidation-specific epitopes and apolipoprotein(a) in progressing and ruptured human coronary and carotid atherosclerotic lesions

“…For example, MB47 detects not only the native apoB-100 moiety of unoxidized LDL but also apoB of minimally oxidized LDL and even apoB fragments of extensively oxidized LDL. MDA2 can recognize both MDA-LDL as well as MDA-lysine epitopes on other proteins in the vessel wall, such as apoAI ( 28,29 ), and it has also been utilized for noninvasive imaging of experimental atherosclerotic lesions (3)(4)(5). E06 is a well-characterized murine IgM natural antibody cloned from apoE Ϫ / Ϫ mice ( 20 ) that has previously been used to stain mouse and rabbit atherosclerotic lesions and to image atherosclerotic lesions in apoE Ϫ / Ϫ mice using magnetic resonance techniques (3)(4)(5).…”

“…IK17 is a human Fab fragment derived from a phage display library, and it binds to a unique MDA-like epitope present on both MDA-LDL and copper-oxidized LDL. IK17 has also been used in detecting and imaging atherosclerosis in LDLR Ϫ / Ϫ mice and apoE Ϫ / Ϫ mice (3)(4)(5)22 ).…”

“…The PC is preserved in this setting and is the moiety recognized by E06. E06 recognizes a variety of OxPL with varying sn2 chain lengths terminated by aldehydes, such as 5,6, and even 9 carbon lengths. It would not recognize oxovaleryl bound to protein unless it was present as the sn2 side chain of an OxPL ( 19,20 ).…”

Adaptive immunity in atherogenesis: new insights and therapeutic approaches

“…Noninvasive magnetic resonance imaging of OSEs is accomplished by injection of these nanoparticles and imaging of the abdominal aorta in cholesterol-fed Apoe -/-mice. Note the lack of signal at the preinjection scan and the strong signal (white contrast) in the 48-hour scan (156). Arrow indicates lesions in the abdominal aorta.…”

“…Importantly, peritoneal macrophages isolated from Adv-IK17-scFv-treated mice had decreased lipid accumulation, consistent with the ability of IK17 to inhibit oxLDL uptake by macrophages. Images reprinted with permission from Biomarkers in Medicine (154), Arteriosclerosis, Thrombosis, and Vascular Biology (155), and Journal of the American College of Cardiology (156).…”