Animal model and study protocol

Autoimmune myocarditis was induced as previously described.6 Nine male Lewis rats aged 6 to 8 weeks (weight, 302.2 ± 17.3 g) were twice immunized with subcutaneous injections of 5 mg/mL pig cardiac myosin (M0531; Sigma Aldrich) in an equal volume of complete Freund’s adjuvant supplemented with 1 mg/mL mycobacterium tuberculosis (F5881; Sigma Aldrich). The two inoculations were given 7 days apart into the hock of the left foot. An intraperitoneal injection of 250 ng/mL pertussis toxin (P2980; Sigma Aldrich) was also given at the time of the first inoculation to enhance immunization. Six control rats (weight, 331.0 ± 9.5 g) twice received the adjuvant alone. All procedures were carried out under isoflurane anesthesia (1.5% to 2.5%). For analgesia, buprenorphine (0.03 mg/kg) was administered two times a day for 2 days after immunization.

PET imaging with [68Ga]Ga-DOTA-Siglec-9 and contrast-enhanced CT were performed on day 21 after the first immunization. To localize the myocardium, contrast-enhanced CT was performed immediately after the PET scan. In three immunized rats, an [18F]FDG scan was acquired 1 day before the [68Ga]Ga-DOTA-Siglec-9 injection to further facilitate the localization of the myocardium. A dose of 1 mg/kg of anti-VAP-1 polyclonal antibody was injected intravenously 10 minutes before sacrifice into six immunized rats and two controls to allow detection of luminal VAP-1 by immunofluorescence. The imaged rats were euthanized at 70 minutes post-injection of [68Ga]Ga-DOTA-Siglec-9, blood was drawn by cardiac puncture, and the heart and other organs were excised, weighed, and measured for radioactivity to determine tracer biodistribution using a gamma counter (Triathler 3″; Hidex).6 The heart was embedded in optimal cutting temperature compound and frozen, and then cut into serial transverse 20 µm and 8 µm cryosections at 1 mm intervals from the base to apex for autoradiography, histology, and immunostaining. A flow diagram of the study protocol and the numbers of rats used is shown in Figure 1.

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

All animal experiments were approved by the national Project Authorization Board (permission number ESAVI/968/04.10.07/2018 and ESAVI/37123/2020) and were carried out in compliance with the EU Directive 2010/EU/63 on the protection of animals used for scientific purposes.

Radiotracers

[18F]FDG was prepared using standard FASTLab® cassettes.22 [68Ga]Ga-DOTA-Siglec-9 was prepared according to previously described procedures.14,18 Briefly, a cyclic peptide, CARLSLSWRGLTLCPSK, with disulfide bridged cysteines consisting of residues 283 to 297 from the Siglec-9 and 8-amino-3,6-diooxaoctanoyl linker (polyethylene glycol derivative) between DOTA chelator and peptide (Peptide Specialty Laboratories GmbH), was labeled with 68Ga. The radiochemical purity determined by reversed-phase radio-HPLC was > 95% and the molar activity was 25.8 ± 8.3 GBq/µmol.

The [68Ga]Ga-DOTA-control peptide was created from the cyclic [68Ga]Ga-DOTA-Siglec-9 peptide CARLSLSWRGLTLCPSK by first mutating Arg3, Trp8, and Arg9 to alanines. These residues were selected since the double mutated R3A/R9A peptide does not bind human VAP-1 (hVAP-1).20 Trp8 was additionally mutated to alanine since its predicted binding site is not conserved in human VAP-1 and rat VAP-1.23 Thereafter, the residues in the resulting CAALSLSAAGLTLCPSK peptide were scrambled manually by avoiding Siglec-9-derived amino acid repeats and by distributing polar residues throughout the sequence to avoid hydrophobic patches. The constructed [68Ga]Ga-DOTA-control, CSALATGLSALALCPSK, the R3A/R9A, and the [68Ga]Ga-DOTA-Siglec-9 peptides were docked into the homology model for rat VAP-1 (rVAP-1) with Gold24 using the pyridazinone inhibitor in the crystal structure of hVAP-1 (Protein Data Bank (PDB) ID 4bty)25 as the center of the binding site. The rVAP-1 model was created with Modeller26 using the published sequence alignment23 and hVAP-1 structure (PDB ID 4bty). Additionally, all peptides were docked into hVAP-1 for comparison. Docking of the peptides to hVAP-1 and rVAP-1 resulted in 10 binding modes for each peptide. Trp8 in the [68Ga]Ga-DOTA-Siglec-9 peptide binds close to the specificity pocket of hVAP-1 (Asp180, Thr210, and Leu177).23,25 This binding site is not conserved in rVAP-1 where the corresponding residues are Gln180, Lys210, and Gln177.23 Trp8 in the docked [68Ga]Ga-DOTA-Siglec-9 and R3A/R9A peptides binds near Gln180 and Gln177 in rVAP-1, whereas the constructed 68Ga-DOTA-control with scrambled sequence and without any tryptophanes does not interact with these residues (Supplemental Figure 1).

In vivo PET/CT imaging

A small animal PET/CT scanner (Inveon Multimodality; Siemens Medical Solutions) was used to perform in vivo imaging with [68Ga]Ga-DOTA-Siglec-9 and [18F]FDG in the rats. The rats were injected with 50.4 ± 2.3 MBq [68Ga]Ga-DOTA-Siglec-9 via the tail vein. Sixty-minute dynamic PET acquisitions (6 × 10 seconds, 4 × 60 seconds, 5 × 300 seconds, and 3 × 600 seconds time frames) were acquired from four immunized rats to study the kinetics of the tracer. Thirty-minute static PET starting at 30 minutes post-injection was then acquired from the rest of the rats. The PET data were reconstructed using an iterative three-dimensional ordered subset expectation maximization using maximum a priori with shifted Poisson distribution (OSEM3D/SP-MAP) algorithm with 2 OSEM iterations and 18 MAP iterations, attenuation and dead time correction, target resolution 1.5 mm, and matrix size 128 × 128.

To visualize myocardium, a 40 minute static [18F]FDG (40.4 ± 1.6 MBq) PET acquisition starting at 20 minutes post-injection was performed the day before the [68Ga]Ga-DOTA-Siglec-9 study in three immunized rats. In addition, 300 μL of intravascular iodinated contrast agent (eXia™ 160XL; Binitio Biomedical Inc.) was injected into all rats to acquire high-resolution CT imaging immediately after the [68Ga]Ga-DOTA-Siglec-9 PET, as described in a previous study.18

Uniformly sized regions of interest (ROIs) were defined in the left ventricle (LV) myocardium in the [68Ga]Ga-DOTA-Siglec-9 images, which were co-registered with the CT or [18F]FDG uptake maps using Carimas 2.9 software (Turku PET Centre). These ROIs were based on macroscopically defined inflammation in the hematoxylin and eosin (H&E) stained sections, with or without visually increased in vivo [68Ga]Ga-DOTA-Siglec-9 uptake. ROIs were also defined in the septal wall to measure tracer uptake in the myocardium outside lesions. The mean standardized uptake value (SUVmean) was compared between histologically confirmed myocardial lesions and myocardium outside lesions. Additional ROIs were defined in the lung, liver, kidney, foreleg muscle, LV cavity, and vena cava (blood). In rats for which dynamic datasets were acquired, decay-corrected time-activity curves and myocardial lesion-to-blood (average of LV and vena cava) uptake ratio curves were determined.

Ex vivo autoradiography, histology, and immunostaining

Ex vivo digital autoradiography was performed on 20 μm cryosections of the myocardium of the imaged rats with [68Ga]Ga-DOTA-Siglec-9, as previously described.14,18 ROIs were defined in myocardial lesions and myocardium outside lesions on the basis of H&E staining of the same section, and the results are presented as average photo-stimulated luminescence per square millimeter (PSL/mm2).

For general histology, H&E staining was performed on 20 μm cryosections. For immunohistochemistry, 8 μm cryosections were stained with a monoclonal mouse anti-rat CD68 antibody (1:1000, MCA341GA, Bio-Rad) to detect macrophages, a polyclonal CD31 antibody (1:200, NB100-2284, Novus Biologicals) to detect vascular endothelium, and monoclonal mouse anti-actin α-smooth muscle antibody (1:12,000, A5228-200, Sigma Aldrich) to detect α-smooth muscle actin (α-SMA) in myofibroblasts with appropriate peroxidase conjugated second stage antibodies. 3,3′-Diaminobenzidine (DAB) was used as a chromogen. Immunofluorescence staining with anti-VAP-1 antibody was performed on 8 μm cryosections to detect VAP-1, as previously described.14 The heart cryosections of the rats that received intravenous anti-VAP-1 antibody (clone 174-5, 1 mg/kg diluted in saline) 10 minutes before sacrifice (five immunized and two controls) were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:100, Sigma F28883) + 5% normal rat serum followed by Alexa Fluor® 488 conjugated anti-FITC (5 mg/mL Invitrogen A11029) and Hoechst (1:5000, Thermo Scientific 62249). The heart cryosections of the other rats (four immunized and four controls) were incubated with polyclonal anti-VAP-1 antibody,14 followed by Alexa Fluor® 488 conjugated goat anti-rabbit IgG secondary antibody (Life Technologies, A11034).

Additional H&E and immunohistochemical staining with a monoclonal mouse anti-rat CD68 antibody (1:2000, MCA341GA, Bio-Rad) and a polyclonal anti-VAP-1 antibody14 were performed on 4 µm paraffin-embedded sections and 8 μm cryosections of thymus, white adipose tissue, spleen, and bone marrow of immunized rats that received intravenous anti-VAP-1 antibody (clone 174-5, 1 mg/kg diluted in saline) 10 minutes before sacrifice.

Human myocardial samples

Two myocardial autopsy samples from two patients who died from cardiac sarcoidosis and one myocardial sample of a heart explanted because of sarcoidosis were cut into serial 4-μm paraffin sections. Use of human samples was approved by hospital ethical review board (HUS317/13/03/01/2015 and HUS/1068/2016), the National Authority for Medicolegal Affairs (4615/06.01.03.01/2016), and the National Institute for Health and Welfare (THL/691/5.05.00/2016).27

H&E staining was performed for general histology. Monoclonal mouse anti-human CD68 antibody (1:200, M0876, Dako Agilent Technologies Inc.) and polyclonal anti-VAP-1 antibody produced in rabbits against recombinant human VAP-128 were used to detect macrophages and VAP-1, respectively, in myocardial lesions.

Studies with [68Ga]Ga-DOTA-control peptide

An in vivo assessment of [68Ga]Ga-DOTA-control peptide binding was performed in four immunized rats (weight, 298.7 ± 9.5 g). The rats were injected with 40.8 ± 5.8 MBq [68Ga]Ga-DOTA-control peptide via the tail vein and were euthanized at 70 minutes post-injection, after which biodistribution analysis and ex vivo autoradiography were performed on 20 μm cryosections of the myocardium.

In vitro binding of [68Ga]Ga-DOTA-Siglec-9

To further evaluate the specificity of the tracer, in vitro binding of [68Ga]Ga-DOTA-Siglec-9 in myocardial tissue sections from three immunized rats was studied with or without 10 minutes pre-incubation with a 100-fold molar excess of non-labeled DOTA-Siglec-9 peptide as a blocker. Total and blocked binding of [68Ga]Ga-DOTA-Siglec-9 was assessed by digital autoradiography.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Unpaired or paired t tests were applied to test single comparisons between normally distributed data to evaluate differences in [68Ga]Ga-DOTA-Siglec-9 uptake in healthy myocardium (control rats), myocardial lesions, and myocardium outside lesions. Mann–Whitney U test and Kolmogorov–Smirnov test were applied to single comparisons between non-normally distributed data. Normality distribution assumption was checked with Shapiro–Wilk test, and assumption of variance equality was checked with Fisher’s F test. GraphPad Prism 6 Software was used to perform the analyses, and statistical significance was set at P < .05.