Evaluation of [99mTc]Radiolabeled Macrophage Mannose Receptor-Specific Nanobodies for Targeting of Atherosclerotic Lesions in Mice
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Macrophage accumulation characterizes the development of atherosclerotic plaques, and the presence of certain macrophage subsets might be an indicator of plaque phenotype and (in)stability. The macrophage mannose receptor (MMR) is expressed on alternatively activated macrophages and found at sites of intraplaque hemorrhage and neovascularization. It has been proposed as target to identify vulnerable plaques. Therefore, we aimed to assess the feasibility of using anti-MMR nanobodies (Nbs) as molecular tracers for nuclear imaging in an animal model of atherosclerosis.
Anti-MMR and control Nb, radiolabeled with Tc-99m, were injected in ApoE−/− and/or C57Bl/6 mice (n = 6). In vivo competition studies involving pre-injection of excess of unlabeled anti-MMR Nb (n = 3) and injection of anti-MMR Nb in MMR−/− mice (n = 3) were performed to demonstrate specificity. At 3 h p.i. radioactive uptake in organs, tissues and aorta segments were evaluated. Autoradiography and immunofluorescence were performed on aortic sections.
Significantly higher uptake was observed in all aortic segments of ApoE−/− mice injected with anti-MMR Nb compared to control Nb (1.36 ± 0.67 vs 0.38 ± 0.13 percent of injected dose per gram (%ID/g), p ≤ 0.001). Surprisingly, high aortic uptake was also observed in C57Bl/6 mice (1.50 ± 0.43%ID/g, p ≥ 0.05 compared to ApoE−/−), while aortic uptake was reduced to background levels in the case of competition and in MMR−/− mice (0.46 ± 0.10 and 0.22 ± 0.06%ID/g, respectively; p ≤ 0.001). Therefore, expression of MMR along healthy aortas was suggested. Autoradiography showed no specific radioactive signal within atherosclerotic plaques, but rather localization of the signal along the aorta, correlating with MMR expression in perivascular tissue as demonstrated by immunofluorescence.
No significant uptake of MMR-specific Nb could be observed in atherosclerotic lesions of ApoE−/− mice in this study. A specific perivascular signal causing a non-negligible background level was demonstrated. This observation should be considered when using MMR as a target in molecular imaging of atherosclerosis, as well as use of translational animal models with vulnerable plaques.
Key WordsNanobody Atherosclerosis Macrophage mannose receptor Molecular imaging
We thank Cindy Peleman for her technical assistance.
This research has been supported by a grant of Research Foundation—Flanders (FWO), Scientific Fund Willy Gepts—UZ Brussel, Strategic Basic Research (Strategisch Basis Onderzoek—Inflammatrack (SBO)), and Industrial Research Fund (Industrieel OnderzoeksFonds (IOF)).
Compliance with Ethical Standards
Conflict of Interest
Drs. Henri Baudhuin, Isabel Remory, Kris Gillis, Pieterjan Debie, Ahmet Krasniqi, and Bernard Cosyns declare that they have no conflict of interest. Vrije Universiteit Brussel (including Drs. Gezim Bala, Tony Lahoutte, Geert Raes, Nick Devoogdt, and Sophie Hernot) has a patent issued (US20160024213A1) regarding anti-macrophage mannose receptor single variable domains for use in cardiovascular diseases.
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