Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis

Figure 1. A flow diagram of the study design and the numbers of animals used. *Four had a dynamic scan and the others a static scan. **A dose of 1 mg/kg of anti-VAP-1 polyclonal antibody was injected intravenously 10 minutes before sacrifice to detect surface-bound VAP-1 by immunofluorescence

Figure 2. (A) A myocardial lesion from a rat with autoimmune myocarditis identified by hematoxylin and eosin (H&E) staining. A consecutive cryosection stained with immunofluorescence shows intracellular and surface-bound vascular adhesion protein-1 (VAP-1). Rat received intravenous anti-VAP-1 antibody 10 minutes before sacrifice and staining was performed using only the secondary antibody. There are numerous CD68-positive macrophages in the inflammatory lesion as well as α-smooth muscle actin (α-SMA) on myofibroblasts. Staining with antibody against CD31 indicates the presence of vascular endothelial cells. (B) Histology and immunohistochemical and immunofluorescence staining of a control rat myocardium

Figure 3. Vascular adhesion protein-1 (VAP-1) is detected in a human coronary artery smooth muscle cells and in the endothelial cells of capillaries. Black arrow in hematoxylin and eosin (H&E) staining indicates the artery inside a sarcoid granuloma. Granuloma forming giant cells and macrophages around the artery (black arrow) are highlighted in CD68-staining. VAP-1 staining of consecutive section shows intensive VAP-1-staining in arterial medial smooth muscle cells and less intense staining in the capillary endothelial cells

Figure 4. (A) Representative in vivo PET images with [18F]FDG and [68Ga]Ga-DOTA-Siglec-9 tracers in a rat with autoimmune myocarditis. PET images at 30 to 60 minutes show focal [68Ga]Ga-DOTA-Siglec-9 uptake (white arrows) in the posterior left ventricle (LV) wall. (B) Ex vivo autoradiography of [68Ga]Ga-DOTA-Siglec-9 uptake shows co-localization with edematous and hemorrhagic myocardial lesion in the posterior wall of the LV, (C) which is confirmed by consecutive sections stained with hematoxylin and eosin (H&E)

Figure 5. Quantification of [68Ga]Ga-DOTA-Siglec-9 PET imaging. (A) Time-activity curves from rats with autoimmune myocarditis (n = 4) show that [68Ga]Ga-DOTA-Siglec-9 uptake (mean standardized uptake value, SUVmean) remains slightly higher in the myocardial lesions than in the blood (average of inferior vena cava and left ventricle). Bars indicate standard deviation. Note that the y-axis has a logarithmic scale. (B) The average myocardial [68Ga]Ga-DOTA-Siglec-9 uptake (SUVmean) 30 to 60 minutes after injection is higher in the myocardium of immunized rats than in the myocardium of control rats (n = 6). (C) Bars demonstrate [68Ga]Ga-DOTA-Siglec-9 uptake by ex vivo autoradiography as mean photo-stimulated luminescence per square millimeter (PSL/mm2) in myocardial lesions and myocardium outside lesions of immunized rats (n = 8), and myocardium of control rats (n = 6). Values are mean ± SD; unpaired t test for comparison of immunized rats’ myocardium and control myocardium, and paired t test for comparison of myocardial lesions and myocardium outside lesions

Figure 6. Hematoxylin and eosin (H&E), CD68 macrophage, and VAP-1 immunohistochemical staining of (A) spleen and (B) bone marrow cryosections from a rat with autoimmune myocarditis. Rat received intravenously anti-VAP-1 antibody 10 minutes before sacrifice and staining was performed using only the secondary antibody. H&E and CD68 macrophage staining of paraffin-embedded sections of (C) thymus and (D) white adipose tissue from a rat with autoimmune myocarditis