In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe^−/−mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using ¹⁸F-fluorothymidine and ¹⁸F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe^−/− mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.

Results from nanoScan® PET/CT

• The authors observed compensatory higher 18F-FDG uptake in the remote myocardium of mice with permanent coronary artery occlusion, compared with mice with IR injury and control mice (Figure 1E.)
• 89Zr-labeled CD11b-specific nanobody PET imaging detected 3-fold higher uptake in the infarct zones of IR and PO mice compared with the same cardiac region of noninfarcted mice (Figure 2E)
• ⁶⁴Cu-DOTA-ECL1i was used to image CCR2 expression. CCR2 PET imaging of the two myocardial infarction models revealed increased signal in the infarct area of mice subjected to PO compared with control mice (Figure 2H)
• ¹⁸F-FLT uptake in the bone marrow was significantly higher in both myocardial infarction mouse models compared with control animals, but there was no difference between the two models (Figures 3C and D)

Figure 1. IR and PO Result in Distinct Myocardial Injury and Cardiac Function Profiles

(A) This study compared mouse models of ischemia -reperfusion (IR) injury, with 40 minutes of ischemia, and permanent left anterior descending coronary artery occlusion (PO). (B) We performed 18F-fluorodeoxyglucose (FDG) PET imaging 2 days after IR or PO surgery. Late gadolinium enhancement (LGE) cardiac MRI was performed 1 day after IR or PO surgery, followed by survival analysis for 1 week. (C) FDG PET/CT 2 days after surgery allowed accurate delineation of the infarct zone. Dashed lines highlight the infarct area. (D) 18F-FDG uptake as mean standardized uptake values (SUVs) of the infarct area and as % injected dose (ID)/g infarct tissue (n = 5-6; P = 0.004 for SUVmean; P = 0.002 for %ID/g; 2-tailed Mann-Whitney test). (E) 18F-FDG uptake in the remote myocardium of mice with PO and IR and the corresponding healthy myocardium of control mice (SUVmean: n = 5-7, P = 0.001 control vs PO, P = 0.132 IR vs PO; %ID/g: n = 5-8, P = 0.025 IR vs PO, P < 0.001 control vs PO; Kruskal-Wallis test). (F) Representative LGE cardiac MRI; arrows indicate infarct area. (G) LGE area in % of left ventricle (n = 9-14; P < 0.001; 2-tailed Student’s t-test). (H) Left ventricular ejection fraction (LVEF) in % for control, IR, and PO mice (n = 9-14; P < 0.001 for IR vs PO, ctrl vs IR, control vs PO; 1-way ANOVA). (I) Simple survival analysis for IR and PO mice for 1 week after myocardial infarction induction (n = 26-29; log-rank test: P = 0.015). Data are presented as mean ± SEM, unless otherwise specified. ∗P <0.05; ∗∗P <0.01; ∗∗∗P <0.001. ANOVA = analysis of variance; MRI =magnetic resonance imaging.

Figure 2. Ischemia Severity Dictates Myeloid Cell Influx Into the Ischemic Myocardium

(A) Myeloid cell influx to the ischemic myocardium assessed by multimodal imaging. (B) 19F-MRI of the heart 2 days after IR/PO surgery and 1 day after PERFECTA nanotracer injection. (C) 19F concentration in the infarct zone of PO mice compared with IR and control mice, expressed as target-to-background ratio (TBR; n = 5-8; P = 0.021 for control vs IR; P < 0.001 for ctrl vs PO; P = 0.22 IR vs PO; Kruskal-Wallis test). Fused 19F-MRI with fluorine uptake in the infarct zone. (D) CD11b PET was performed 3 days after IR/PO surgery and 2 days after tracer injection. (E) CD11b PET signal quantification expressed as SUVmean (n = 5-8; P = 0.002 control vs IR; P = 0.002 control vs PO; P = 0.91 IR vs PO; Kruskal-Wallis test) and fused PET/CT images of the heart. (F) Ex vivo quantification showed higher tracer uptake for mice with IR and PO injury compared to control mice (n = 6-8; P = 0.019 control vs IR; P < 0.001 control vs PO; P = 0.14 IR vs PO; Kruskal-Wallis test). (G) CCR2 PET to image inflammatory monocytes was performed 2 days after IR/PO surgery. The scan occurred during the first 60 minutes after 64Cu-DOTA-ECL1i injection. (H) 64Cu-DOTA-ECL1i PET signal quantification expressed as SUVmean (n = 7-9; P = 0.083 control vs IR; P = 0.021 control vs PO; P = 0.64 IR vs PO; Kruskal-Wallis test) and representative images of control, IR, and PO mice. (I) Ex vivo quantification of tracer uptake by gamma-counting (n = 7; P = 0.011 control vs IR; P < 0.001 control vs PO; P = 0.21 IR vs PO; Kruskal-Wallis test). (J) Flow cytometry of the infarct zone 2 days after myocardial infarction. (Left) Representative flow plots for control, IR and PO hearts, gated on leukocytes. (Right) Quantification of CD11b+/Ly6G- cells (n = 9-14; P < 0.001 control vs IR; P < 0.001 control vs PO; P = 0.092 IR vs PO; Kruskal-Wallis test), neutrophils (n = 9-14; P = 0.002 control vs IR; P < 0.001 control vs PO; P = 0.028 IR vs PO; Kruskal-Wallis test), Ly6Chigh monocytes (n = 9-14; P = 0.001 control vs IR; P < 0.001 control vs PO; P = 0.069 IR vs PO; Kruskal-Wallis test), and macrophages (n = 9-14; P < 0.001 control vs IR; P < 0.001 control vs PO; P = 0.87 IR vs PO; Kruskal-Wallis test) in the infarct. Data are presented as mean ± SEM, unless otherwise specified. ∗P <0.05; ∗∗P <0.01; ∗∗∗P <0.001. Abbreviations as in Figure 1.

Figure 3. Bone Marrow Activity After Cardiac Ischemia-Reperfusion and Permanent Left Anterior Descending Coronary Artery Occlusion Are Similar

(A) Cardiac ischemia induces proliferation, metabolic activation, and myeloid cell egress in/from the bone marrow. We probed these processes via molecular imaging. (B) 18F-fluorothymidine (FLT) PET/CT was performed 2 days after IR/PO surgery. 18F-FLT was injected 90 minutes before the scan. (C) Quantification of 18F-FLT signal in the lumbar vertebrae (n = 5-7; P = 0.032 control vs IR; P = 0.018 control vs PO; P = 0.92 IR vs PO; Kruskal-Wallis test) and representative 18F-FLT PET/CT images showing higher activity for both IR and PO mice. (D) Ex vivo gamma counting of 18F-FLT uptake in the bone marrow (n = 8-9; P = 0.006 control vs IR; P = 0.006 control vs PO; P = 0.92 IR vs PO; Kruskal-Wallis test). (E) Flow cytometry analysis of bone marrow cells 3 days after IR/PO. Representative histograms of BrdU expression in Lin−Sca-1+cKit+ (LSK) cells and quantification of BrdU-positive cells. BrdU (1 mg) was injected i.P. 24 hours before analysis (n = 6-8, P = 0.014 control vs IR; P < 0.001 control vs PO; P = 0.18 IR vs PO; Kruskal-Wallis test). (F) 18F-FDG PET/CT was performed 2 days after IR/PO surgery. 18F-FDG was injected 30 minutes before imaging. (G) Quantification of 18F-FDG signal in the lumbar vertebrae (n = 5-7; P = 0.005 control vs IR; P = 0.025 control vs PO; P = 0.52 IR vs PO; Kruskal-Wallis test) and representative images showing higher activity in IR and PO mice compared with control mice. (H) Ex vivo gamma counting of 18F-FDG uptake in the bone marrow (n = 6-8; P = 0.008 control vs IR; P = 0.011 control vs PO; P = 0.92 IR vs PO; Kruskal-Wallis test). (I) Number of LSK cells in the bone marrow 3 days after IR/PO (n = 6-8; P = 0.003 control vs IR; P = 0.052 control vs PO; P = 0.33 IR vs PO; Kruskal-Wallis test) with representative plots. (J) CD11b PET was performed 3 days after myocardial infarction. The 89Zr-labeled CD11b-specific nanobody was injected 2 days after myocardial infarction. (K) Quantification of PET signal in the lumbar vertebrae (n = 5-8; P = 0.001 control vs IR; P = 0.032 control vs PO; P = 0.41 IR vs PO; Kruskal-Wallis test) and representative images showing lower signal in mice with IR or PO surgery compared with control. (L) Ex vivo gamma counting of tracer uptake in the bone marrow (n = 6-8; P = 0.017 control vs IR; P = 0.039 control vs PO; P = 0.77 IR vs PO; Kruskal-Wallis test). (M) Representative flow plots of blood samples, gated on viable cells. Significantly higher numbers of CD11b+ cells were observed in the blood of IR and PO animals 2 days after infarction (n = 9-14; P = 0.021 control vs IR; P = 0.025 control vs PO; P = 0.87 IR vs PO; 1-way ANOVA). Data are presented as mean ± SEM, unless otherwise specified. ∗P <0.05; ∗∗P <0.01, ∗∗∗P <0.001. Abbreviations as in Figures 1 and 2.

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