In vivo Visualization of M2 Macrophages in the Myocardium After Myocardial Infarction (MI) Using 68 Ga-NOTA-Anti-MMR Nb: Targeting Mannose Receptor (MR, CD206) on M2 Macrophages

Abstract

Introduction and objectives: Wound healing after myocardial infarction (MI) is a dynamic and complex multiple phase process, and a coordinated cellular response is required for proper scar formation. The current paradigm suggests that pro-inflammatory monocytes infiltrate the MI zone during the initial pro-inflammatory phase and differentiate into inflammatory macrophages, and then switch their phenotypes to anti-inflammatory during the reparative phase. Visualization of the reparative phase post-MI is of great interest because it may reveal delayed resolution of inflammation, which in turn predicts adverse cardiac remodeling. Imaging of anti-inflammatory macrophages may also be used to assess therapy approaches aiming to modulate the inflammatory response in order to limit MI size. Reparative macrophages can be distinguished from inflammatory macrophages by the surface marker mannose receptor (MR, CD206). In this study we evaluated the feasibility of ⁶⁸Ga-NOTA-anti-MMR Nb for imaging of MR on alternatively activated macrophages in murine MI models.

Methods: Wildtype and MR-knockout mice and Wistar rats were subjected to MI via permanent ligation of the left coronary artery. Non-operated or sham-operated animals were used as controls. MR expression kinetics on cardiac macrophages was measured in mice using flow cytometry. PET/CT scans were performed 1 h after intravenous injection of ⁶⁸Ga-NOTA-anti-MMR Nb. Mice and rats were euthanized and hearts harvested for ex vivo PET/MRI, autoradiography, and staining. As a non-targeting negative control, ⁶⁸Ga-NOTA-BCII10 was used.

Results: In vivo-PET/CT scans showed focal radioactivity signals in the infarcted myocardium for ⁶⁸Ga-NOTA-anti-MMR Nb which were confirmed by ex vivo-PET/MRI scans. In autoradiography images, augmented uptake of the tracer was observed in infarcts, as verified by the histochemistry analysis. Immunofluorescence staining demonstrated the presence and co-localization of CD206- and CD68-positive cells, in accordance to infarct zone. No in vivo or ex vivo signal was observed in the animals injected with control Nb or in the sham-operated animals. ⁶⁸Ga-NOTA-anti-MMR Nb uptake in the infarcts of MR-knockout mice was negligibly low, confirming the specificity of ⁶⁸Ga-NOTA-anti-MMR Nb to MR.

Conclusion: This exploratory study highlights the potential of ⁶⁸Ga-NOTA-anti-MMR Nb to image MR-positive macrophages that are known to play a pivotal role in wound healing that follows acute MI.

Keywords: M2 macrophages; PET; inflammation; myocardial infarction; reparative phase.

FIGURE 1

The healing of the myocardium after infarction involves biphasic infiltration and accumulation of monocytes/macrophages to the infarct. Inflammatory monocytes/macrophages accumulate early after infarction and promote digestion and removal of the necrotic tissue, whereas reparative monocytes/macrophages accumulate later and propagate resolution of inflammation, scar formation, and repair. A proper healing process requires a precise balance between removal of debris and regulation of scar formation. Impaired resolution of inflammation (phase II) may result in prolonged inflammatory phase (phase I) and eventually adverse cardiac remodeling.

FIGURE 2

Flow cytometric quantification of (A) total number of macrophages, (B) MR⁺ macrophages, and (C) MR expression level on cardiac macrophages in non-infarcted (steady state) and infarcted (2, 7, and 14 days after coronary ligation) wildtype mice hearts. Data are derived from n = 5-7 mice per time point. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 3

Representative in vivo imaging of ⁶⁸Ga-NOTA-anti-MMR Nb uptake in a longitudinal study. (A) Static PET/CT matched axial, coronal and sagittal slices in the same mouse subjected to coronary ligation and scanned 1 h after injection of ⁶⁸Ga-NOTA-anti-MMR Nb (2, 7, and 14 days after MI) and ¹⁸F-FDG (7 days after MI). In order to validate the results obtained by in vivo PET/CT imaging (in n = 6 mice) and to confirm the cardiac origin of the in vivo signal, hearts from two MI-induced mice were also scanned ex vivo. (B) MR only and (C) PET/MRI of the heart excised from a mouse, 7 days after coronary ligation, confirming augmented ⁶⁸Ga-NOTA-anti-MMR Nb uptake in the area close to the ligation site.

FIGURE 4

Representative ⁶⁸Ga-NOTA-anti-MMR Nb PET/CT (A) coronal and (B) axial images. In order to validate the results obtained by in vivo PET/CT imaging and to confirm the cardiac origin of the in vivo signal (n = 7), hearts from two MI-induced rats were also scanned ex vivo. (C) Photograph, (D) MRI only and (E) PET/MRI of the representative heart excised from rat, 7 days after coronary ligation, confirmed an augmented ⁶⁸Ga-NOTA-anti-MMR Nb uptake in the area close to the ligation site. ⁶⁸Ga-NOTA-anti-MMR Nb exhibited elevated uptake in scars from operation (blue arrowheads).

FIGURE 5

Binding specificity test in rats. (A) In vivo (PET/CT) and ex vivo (autoradiography) evaluation of targeting ⁶⁸Ga-NOTA-anti-MMR Nb and non-targeting ⁶⁸Ga-NOTA-BCII10 Nb biodistribution in infarcted (7 days after coronary ligation) and non-infarcted (sham-operated) rat hearts and corresponding H.E. staining. (B) PET image-derived and (C) autoradiography image-derived infarct-to-remote area uptake ratios derived from n = 4 MI rats injected with ⁶⁸Ga-NOTA-anti-MMR Nb and n = 4 MI rats injected with ⁶⁸Ga-NOTA-BCII10 Nb. QL = quantum level.

FIGURE 6

Binding specificity test in mice. (A) Ex vivo evaluation of ⁶⁸Ga-NOTA-anti-MMR Nb biodistribution in infarcted (7 days after coronary ligation) and non-infarcted (sham-operated) wildtype and MR-knockout mice hearts and corresponding H.E. staining. (B) Autoradiography image-derived infarct-to-remote area uptake ratio (derived from n = 4 MI wildtype and n = 4 MR-knockout mice injected with ⁶⁸Ga-NOTA-anti-MMR Nb). QL = quantum level.

FIGURE 7

Photomicrographs of MR (green) and CD68 (red) immunofluorescence staining. DAPI stained nuclei are shown in blue. Overlapping domains of MR and CD68 expression are shown in yellow. A portion of CD68⁺ macrophages located in the 7-day-old infarcts excised from rats showed MR expression.