Molecular Imaging and Biology

, Volume 20, Issue 2, pp 260–267 | Cite as

Evaluation of [99mTc]Radiolabeled Macrophage Mannose Receptor-Specific Nanobodies for Targeting of Atherosclerotic Lesions in Mice

  • Gezim Bala
  • Henri Baudhuin
  • Isabel Remory
  • Kris Gillis
  • Pieterjan Debie
  • Ahmet Krasniqi
  • Tony Lahoutte
  • Geert Raes
  • Nick Devoogdt
  • Bernard Cosyns
  • Sophie Hernot
Research Article



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 Words

Nanobody 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.


  1. 1.
    Quillard T, Libby P (2012) Molecular imaging of atherosclerosis for improving diagnostic and therapeutic development. Circ Res 111:231–244CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Libby P, Ridker PM, Maseri A (2002) Inflammation and atherosclerosis. Circulation 105:1135–1143CrossRefPubMedGoogle Scholar
  3. 3.
    Libby P (2012) Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol 32:2045–2051CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tarkin JM, Joshi FR, Rudd JHF (2014) PET imaging of inflammation in atherosclerosis. Nat Rev Cardiol 11:443–457CrossRefPubMedGoogle Scholar
  5. 5.
    Colin S, Chinetti-Gbaguidi G, Staels B (2014) Macrophage phenotypes in atherosclerosis. Immunol Rev 262:153–166CrossRefPubMedGoogle Scholar
  6. 6.
    Chinetti-Gbaguidi G, Colin S, Staels B (2015) Macrophage subsets in atherosclerosis. Nat Rev Cardiol 12:10–17CrossRefPubMedGoogle Scholar
  7. 7.
    Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23–35CrossRefPubMedGoogle Scholar
  8. 8.
    Tahara N, Mukherjee J, de Haas HJ et al (2014) 2-Deoxy-2-[18F]fluoro-D-mannose positron emission tomography imaging in atherosclerosis. Nat Med 20:215–219CrossRefPubMedGoogle Scholar
  9. 9.
    Finn AV, Nakano M, Polavarapu R et al (2012) Hemoglobin directs macrophage differentiation and prevents foam cell formation in human atherosclerotic plaques. J Am Coll Cardiol 59:166–177CrossRefPubMedGoogle Scholar
  10. 10.
    Chinetti-Gbaguidi G, Baron M, Bouhlel MA et al (2011) Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways. Circ Res 108:985–995CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Finn AV, Saeed O, Virmani R (2012) Macrophage subsets in human atherosclerosis. Circ Res 110:1–8CrossRefGoogle Scholar
  12. 12.
    Chinetti-Gbaguidi G, Staels B (2012) Response to the letter by Finn et al. Circ Res 110:e65–e66CrossRefGoogle Scholar
  13. 13.
    Kim EJ, Kim S, Seo HS et al (2016) Novel PET imaging of atherosclerosis with 68Ga-labeled NOTA-neomannosylated human serum albumin. J Nucl Med 57:1792–1797CrossRefPubMedGoogle Scholar
  14. 14.
    Kim JB, Park K, Ryu J et al (2016) Intravascular optical imaging of high-risk plaques in vivo by targeting macrophage mannose receptors. Sci Rep 6:22608CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Movahedi K, Schoonooghe S, Laoui D et al (2012) Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages. Cancer Res 72:4165–4177CrossRefPubMedGoogle Scholar
  16. 16.
    Blykers A, Schoonooghe S, Xavier C et al (2015) PET imaging of macrophage mannose receptor-expressing macrophages in tumor stroma using 18F-radiolabeled camelid single-domain antibody fragments. J Nucl Med 56:1265–1271CrossRefPubMedGoogle Scholar
  17. 17.
    Put S, Schoonooghe S, Devoogdt N et al (2013) SPECT imaging of joint inflammation with nanobodies targeting the macrophage mannose receptor in a mouse model for rheumatoid arthritis. J Nucl Med 54:807–814CrossRefPubMedGoogle Scholar
  18. 18.
    Broisat A, Hernot S, Toczek J et al (2012) Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res 110:927–937CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Vaneycken I, Devoogdt N, Van Gassen N et al (2011) Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 25:2433–2446CrossRefPubMedGoogle Scholar
  20. 20.
    Bala G, Blykers A, Xavier C et al (2016) Targeting of vascular cell adhesion molecule-1 by 18F-labelled nanobodies for PET/CT imaging of inflamed atherosclerotic plaques. Eur Hear J - Cardiovasc Imaging 17:1001–1008CrossRefGoogle Scholar
  21. 21.
    De Vos J, Mathijs I, Xavier C et al (2014) Specific targeting of atherosclerotic plaques in ApoE−/− mice using a new camelid sdAb binding the vulnerable plaque marker LOX-1. Mol Imaging Biol 16:690–698CrossRefPubMedGoogle Scholar
  22. 22.
    Zheng F, Devoogdt N, Sparkes A et al (2015) Monitoring liver macrophages using nanobodies targeting Vsig4: concanavalin A induced acute hepatitis as paradigm. Immunobiology 220:200–209CrossRefPubMedGoogle Scholar
  23. 23.
    Keyaerts M, Xavier C, Heemskerk J et al (2016) Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. J Nucl Med 57:27–33CrossRefPubMedGoogle Scholar
  24. 24.
    Xavier C, Devoogdt N, Hernot S et al (2012) Site-specific labeling of his-tagged Nanobodies with 99mTc: a practical guide. Methods Mol Biol 911:485–490CrossRefPubMedGoogle Scholar
  25. 25.
    Stöger JL, Gijbels MJJ, van der Velden S et al (2012) Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis 225:461–468CrossRefPubMedGoogle Scholar
  26. 26.
    Schwartz CJ, Mitchell JR (1962) Cellular infiltration of the human arterial adventitia associated with atheromatous plaques. Circulation 26:73–78CrossRefPubMedGoogle Scholar
  27. 27.
    Linehan SA, Martínez-Pomares L, Stahl PD, Gordon S (1999) Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: in situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells. J Exp Med 189:1961–1972CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lardenoye JH, Delsing DJ, de Vries MR et al (2000) Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res 87:248–253CrossRefPubMedGoogle Scholar
  29. 29.
    Zhang S, Picard MH, Vasile E et al (2005) Diet-induced occlusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction, and premature death in scavenger receptor class B type I-deficient, hypomorphic apolipoprotein ER61 mice. Circulation 111:3457–3464CrossRefPubMedGoogle Scholar
  30. 30.
    Van der Donckt C, Van Herck JL, Schrijvers DM et al (2015) Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death. Eur Heart J 36:1049–1058CrossRefPubMedGoogle Scholar

Copyright information

© World Molecular Imaging Society 2017

Authors and Affiliations

  • Gezim Bala
    • 1
    • 2
  • Henri Baudhuin
    • 1
  • Isabel Remory
    • 1
    • 3
  • Kris Gillis
    • 1
    • 2
  • Pieterjan Debie
    • 1
  • Ahmet Krasniqi
    • 1
  • Tony Lahoutte
    • 1
    • 4
  • Geert Raes
    • 5
    • 6
  • Nick Devoogdt
    • 1
  • Bernard Cosyns
    • 1
    • 2
  • Sophie Hernot
    • 1
  1. 1.In Vivo Cellular and Molecular Imaging (ICMI/BEFY)Vrije Universiteit BrusselBrusselsBelgium
  2. 2.Centrum voor Hart-en Vaatziekten (CHVZ)UZ BrusselBrusselsBelgium
  3. 3.Department of AnesthesiologyUZBrusselBrusselsBelgium
  4. 4.Nuclear Medicine DepartmentUZ BrusselBrusselsBelgium
  5. 5.Laboratory of Cellular and Molecular Immunology (CMIM)Vrije Universiteit BrusselBrusselsBelgium
  6. 6.Myeloid Cell Immunology Lab, VIB Inflammation Research CenterGhentBelgium

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