Macrophage mannose receptor CD206-targeted PET imaging in experimental acute myocardial infarction

Abstract

Background: The macrophage mannose receptor (CD206) is expressed predominantly on the surface of M2-type macrophages, which play a role in resolution of inflammation after myocardial injury. The purpose of this study was to evaluate the utility of CD206-targeted PET tracer Al[¹⁸F]F-NOTA-D10CM, a fluorinated mannosylated dextran derivative, for imaging immune responses after experimental acute myocardial infarction (MI).

Results: Flow cytometry revealed selective binding of Alexa-488-NOTA-D10CM to human M2-polarized macrophages derived from blood monocytes compared to M1 macrophages. The binding affinity of Al[¹⁸F]F-NOTA-DCM for CD206-positive Chinese hamster ovary cells was 1.83 ± 0.68 nM. In vivo PET and ex vivo autoradiography experiments in Sprague-Dawley rats studied at 3 and 7 days after permanent ligation of the left coronary artery or a sham-operation, showed significantly higher uptake of Al[¹⁸F]F-NOTA-DCM in the MI area than in remote areas, or the myocardium of sham-operated rats. However, there was no difference in uptake in the MI area between day 3 and day 7. Uptake of Al[¹⁸F]F-NOTA-DCM in the MI area correlated positively with the area-% of CD206-positive staining of the left ventricular myocardium (r = 0.481, P = 0.006). In vitro competition studies on tissue cryosections using a molar excess of unlabeled D10CM revealed a reduction of approximately 85%, confirming specific tracer binding.

Conclusion: Al[¹⁸F]F-NOTA-D10CM PET detects overexpression of CD206 after ischemic myocardial injury, and may be a suitable biomarker for detecting M2-type macrophages associated with the inflammatory process post-MI.

Keywords: CD206; Inflammation; Macrophage mannose receptor; Myocardial infarction; PET.

Fig. 1

Animal study design. Sprague Dawley rats (n = 23) underwent permanent LAD ligation or sham-operation on Day 0. Then, the rats were divided to four groups: MI Day 3 (Group 1), Sham Day 3 (Group 2), MI Day 7 (Group 3), and Sham Day 7 (Group 4). All rats were imaged with [¹⁸F]FDG PET/CT 1 day before Al[¹⁸F]F-NOTA-D10CM PET/CT. All rats were euthanized immediately after Al[¹⁸F]F-NOTA-D10CM PET/CT imaging to measure ex vivo biodistribution, autoradiography, hematoxylin–eosin (H&E) staining, CD206 immunohistochemical staining, and double CD206 and CD68 immunofluorescence staining of the left ventricle

Fig. 2

Binding of Alexa-488-NOTA-D10CM to human M1 versus M2 macrophages. A Peripheral blood mononuclear cells were polarized into M1 and M2 macrophages in vitro. Representative flow cytometry histograms (upper panels) and quantification (lower panels) of CD206 expression. B Representative flow cytometry histograms (upper panels) and quantification of Alexa-488-NOTA-D10CM binding (lower panels) to M1 and M2 macrophages after a 30-min or 4-h incubation. MFI mean fluorescence intensity. P values are from Student’s t test

Fig. 3

Binding of Al[¹⁸F]F-NOTA-D10CM to CD206⁺ versus CD206⁻ cells. A Representative flow cytometry histograms of mouse CD206 expression on naive Chinese hamster ovary cells (CHO-CD206⁻) and CD206-transfected cells (CHO-CD206⁺), as detected by an AlexaFluor-488 anti-mouse CD206 antibody. B Quantification of CD206 expression. MFI, mean fluorescence intensity. (C) An example of real-time binding of Al[¹⁸F]F-NOTA-D10CM to CHO-CD206⁺ and CHO-CD206⁻ cells, as determined using the LigandTracer instrument. cps counts per second. P values are from Student’s t test

Fig. 4

In vivo PET imaging of rats with myocardial infarction (MI). A Representative Al[¹⁸F]F-NOTA-D10CM and [¹⁸F]FDG PET images of rats on Day 3 and Day 7 after MI. [¹⁸F]FDG visualizes viable myocardium (blue arrows), while Al[¹⁸F]F-NOTA-D10CM shows uptake in the infarcted and border zone areas (red arrows). The white arrows denote tracer uptake in the healing surgical scar. B Time-activity curves of the blood pool, MI area, and remote area on Day 3 and Day 7 after MI, and of the myocardium of sham-operated rats. C Quantification of in vivo Al[¹⁸F]F-NOTA-D10CM PET/CT reveals significantly higher tracer uptake in the MI area both on Day 3 and Day 7 after MI (compared with the remote area or the myocardium of sham-operated rats). SUV standardized uptake value. P values are from one-way ANOVA

Fig. 5

Autoradiography of myocardial Al[¹⁸F]F-NOTA-D10CM uptake and quantification of CD206-positive cells. A Representative Al[¹⁸F]F-NOTA-D10CM autoradiographs, hematoxylin–eosin (H&E) staining, and anti-CD206 immunohistochemical staining of the rat left ventricle on Day 3 and Day 7 after myocardial infarction (MI). Quantification of B Al[¹⁸F]F-NOTA-D10CM autoradiography and C CD206-positive staining reveals significantly higher signals in the infarcted and border zone areas on both Day 3 and Day 7 after MI. D Correlation between the Al[¹⁸F]F-NOTA-D10CM in vivo PET results for the infarct area and A the % CD206-positive stained area or E ex vivo autoradiography of Al[¹⁸F]F-NOTA-D10CM on Day 3 and Day 7 after myocardial infarction. PSL/mm² photostimulated luminescence per square millimeter. P values are from one-way ANOVA and correlation coefficients from Pearson’s analysis

Fig. 6

CD206 and CD68 in rat myocardial tissue after infarction. Double immunofluorescence staining of a rat left ventricle reveals co-localization of CD68-positive staining and macrophage mannose receptor CD206-positive staining in the injured area on Day 7 after MI

Fig. 7

In vitro competitive study confirming Al[¹⁸F]F-NOTA-D10CM binding specificity. A Representative Al[¹⁸F]F-NOTA-D10CM autoradiographs, and hematoxylin–eosin (H&E) and anti-CD206 staining of adjacent cryosections of the left ventricle post-myocardial infarction (MI). B Quantification of Al[¹⁸F]F-NOTA-D10CM binding without and with co-incubation with an excess of unlabeled NOTA-D10CM. PSL/mm² photostimulated luminescence per square millimeter. P values are from Student’s t test