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Introductory Lecture by René Botnar - London, UK
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
Antibiotic treatment with Minocycline affects vascular elastin remodeling distally to the site of injury in a murine model of atherosclerosis (#109)
B. Lavin1, A. Phinikaridou1, M. E. Andia2, M. Potter3, R. M. Botnar1, 2
1 King's College London, School of Biomedical Engineering Imaging Sciences, London, United Kingdom
Vascular interventions aim to treat focal stenosis however, they may trigger systemic responses that accelerate lesions elsewhere. The antibiotic agent minocycline has demonstrated substantial effect on matrix remodeling1-3 and late gadolinium enhancement (LGE) MRI using an elastin-specific contrast agent (Gd-ESMA) can assess matrix remodelling in cardiovascular diseases4-6. We study the merits of Gd-ESMA to assess the impact of aortic injury and minocycline treatment on the plaques located distally to the site of injury in vivo, and whether the response to injury could be driven by monocytes.
Study design is summarized in Fig.1A. Animal surgical model: Aortic injury was performed as described7. In vivo MRI: A 3T MR scanner (Achieva, Philips Healthcare, NL) equipped with a 23mm single-loop microscopy surface coil were used. Images were acquired 2h after intravenous administration of Gd-ESMA (0.2mmol/kg). Acquisition parameters are summarised in Fig.1B. Histology: Atherosclerotic plaques in the brachiocephalic artery (BCA) were analyzed using Verhoeff Van Gieson elastic stain and tropoelastin immunohistochemistry (IHC). FACS: Monocyte count was performed using B220, F4/80, CD155 and Ly6C antibodies.
LGE-MRI images of the BCA (Fig 1C) showed greater enhancement (Fig 1D) and higher R1 relaxation rate (Fig 1E) in the HFD-injury group compared to all other groups, suggesting that vascular injury accelerates atherosclerosis in distal locations compared to HFD alone. Mice treated with minocycline showed decreased Gd-ESMA uptake compared to the untreated groups (Fig 1E-1D). Histology showed increased elastin fibers in the HFD and HFD-injury groups but not in minocycline-treated mice (Fig 1C).
Flow cytometry of monocytes (Fig 2A) showed blood monocytosis in HFD and HFD-injured mice, but not in the minocycline-treated group (Fig 2B). HFD-injured mice showed increased monocyte recruitment to the BCA compared to other groups (Fig 2D). In blood and BCA, a shift from Ly6Chigh to Ly6Clow subtypes was observed in the minocycline-treated mice (Fig 2C-2E). Positive correlation was detected between vessel wall enhancement, R1 and Ly6Chigh monocytes (Fig 2F-2H) measured in the aorta and the BCA.
We demonstrate that focal vascular injury accelerates atherosclerosis in distal vessel segments through a systemic response driven by monocytes. Minocycline treatment alters elastin remodeling and promotes a shift from Ly6Chigh inflammatory to Ly6Clow reparative monocytes retarding intimal thickening distally to the site of injury.
1Ohshima, S. JACC. 2010. 2Shahzad, K. Atherosclerosis. 2011. 3Phinikaridou A. J Am Heart Assoc. 2013. 4Makowski, M.R. Nature Medicine. 2011. 5Botnar, R.M. Circ Cardiovasc Imaging. 2014. 6Protti, A. J Am Heart Assoc. 2015.7Lavin, B. Circ Cardiovasc Imaging. 2015.
The British Heart Foundation (RG/12/1/29262).
(A) Study design. (B) MRI acquisition parameters. (C) 1st row: Fused LGE-MRI/MRA images of the BCA vessel wall; 2nd row: Verhoeff Van Gieson elastic stain of the BCA showing a thicker fibrous cap in the injured groups (arrow); 3rd row: Tropoelastin IHC showing fiber deposition (asterisk) in the HFD and HFD-injury groups. (D) LGE-MRI area and (E) relaxation rate (R1) quantification measured by MRI.
(A) Flow cytometry monocyte gating strategy. (B) Quantification of monocytes (C) and percentage of Ly6Chigh (orange) and Ly6Clow (blue) monocytes in blood. (D) Quantification of monocytes (E) and percentage of Ly6Chigh (orange) and Ly6Clow (blue) monocytes in BCA. LGE-MRI (F), R1 (G) and Ly6Chigh (H) Pearson correlations between aorta and BCA. BCA: Brachiocephalic artery.
Molecular imaging of tropoelastin in plaque progression and instability (#47)
A. Phinikaridou1, S. Lacerda2, B. Lavin1, M. E. Andia3, R. M. Botnar1
1 King's College London, Biomedical Engineering, London, United Kingdom
Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques and has been associated with lesion progression and destabilization [1, 2]. We have developed a tropoelastin-binding gadolinium-based MRI contrast agent (Gd-TESMA) and demonstrated the feasibility of molecular imaging of tropoelastin in mice and rabbits.
Atherosclerotic ApoE-/- mice and New Zealand rabbits were used. Gd-TESMA was synthesized and characterized prior to in vivo use . 3 Tesla MRI: Angiography, delayed-enhanced (DE) MRI and T1 mapping of the brachiocephalic artery (BCA) in mice was performed at 4, 8, and 12 weeks of high-fat-diet and 30min post-injection of Gd-TESMA. Rabbits were imaged two times before and one time after triggering for plaque rupture . Pre-triggered, rabbits were scanned with an elastin-binding contrast agent (Gd-ESMA)(Lantheus Medical Imaging) and then with Gd-TESMA. T1-black-blood (BB), DE-MRI and T1 mapping images were acquired for plaque characterization. Post-triggered images were used to classify plaques in ruptured and stable. Elastin and tropoelastin stainings were used for validation.
DE-MRI (Fig. 1A1-E1 & A2-E2) and R1 maps (Fig. 1A3-E3), post-injection of Gd-TESMA, showed increased vascular enhancement and R1 relaxation rate of the BCA with disease progression and regression after statin-treated treatment (Fig.1E1-3). Histology verified the deposition of tropoelastin fibres (Fig. 1A4-E4, A5-E5, A6-E6). A scrambled probe showed less vascular enhancement compared with the non-scrambled probe (Fig. 1F1-F3). Pre-trigger images of a stable (Fig. 2A-E) and rupture-prone plaques (Fig. 2G-K) detect the lesions. DE-MRI showed vascular enhancement and reduction of T1 relaxation time post-injection of both agents. Post-trigger T1BB images showed thrombus only attached to the ruptured lesion (Fig. 2F, L). Rupture-prone plaques had higher R1 relaxation rate post-injection of Gd-TESMA compared with stable plaques and that allowed their detection with high sensitivity and specificity. Conversely, uptake of Gd-ESMA was similar between the two groups (Fig. 2M-N).
Molecular imaging of tropoelastin allows monitoring of lesion progression and detection of rupture-prone plaque.
1. Krettek, A., et al. ATVB, 2003.
2. Makowski, M., et al., Nat Med, 2011.
3. Phinikaridou, A., et al., ISMRM proceedings, 2016.
4. Phinikaridou, A., et al., Radiol. 2014.
British Heart Foundation
Keywords: atherosclerosis, MRI, tropoelastin, elastin
Targeting malondialdehyde-acetaldehyde epitopes with a human antibody fragment detects clinically relevant atherothrombotic lesions and allows non-invasive PET/MR imaging in experimental models (#119)
M. L. Senders1, 2, X. Que3, Y. S. Cho4, 5, C. Yeang5, H. Groenen1, F. Fay1, A. E. Meerwaldt1, C. Calcagno1, S. Green5, P. Miu5, M. E. Lobatto6, T. Reiner7, Z. A. Fayad1, J. L. Witztum3, W. J. M. Mulder1, S. Tsimikas5, C. Pérez-Medina1
1 Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, New York, United States of America
Oxidation-specific epitopes (OSE) are well defined danger-associated molecular patterns that activate inflammatory pathways leading to initiation and progression of atherosclerosis1,2, the major underlying cause of cardiovascular disease. Despite its tremendous socioeconomic impact, clinical characterization of atherosclerosis remains a challenge, as it mainly relies on the detection of the degree of stenosis3. Here we present a novel positron emission tomography (PET) probe that allows non-invasive imaging of OSE-rich atherosclerotic lesions.
The human monoclonal antigen-binding fragment (Fab) LA25 was identified and characterized after multiple rounds of screening against the oxidation-specific malondialdehyde-acetaldehyde (MAA) epitope. Pharmacokinetics, biodistribution and plaque specificity studies were performed in Apoe-/- mice with Zirconium-89 (89Zr)-labeled LA25. In rabbits, 89Zr-LA25 was used in combination with an integrated clinical PET/magnetic resonance (PET/MR) system. 18F-fluorodeoxyglucose (18F-FDG)-PET and dynamic contrast-enhanced MR imaging (DCE-MRI) were used to evaluate vessel wall inflammation and plaque neovascularization, respectively. Extensive ex vivo validation was carried out by a combination of gamma counting, near-infrared fluorescence, autoradiography, immunohistochemistry, and immunofluorescence.
LA25 bound specifically to MAA epitopes in advanced and ruptured human atherosclerotic plaques with accompanying thrombi and in debris from distal protection devices. In Apoe-/- mice, 89Zr-LA25 accumulation in the aorta was significantly higher than for 89Zr-LA24, a non-targeted Fab that was used as chemical control (Fig. 1B-C). Analysis of aortic root sections revealed extensive co-localization of 89Zr-LA25 radioactivity to macrophage-rich areas (Fig. 1E). PET/MR imaging 24 hours after injection of 89Zr-LA25 showed increased uptake in the abdominal aorta of atherosclerotic rabbits compared to non-atherosclerotic controls, confirmed by ex vivo gamma counting (P=0.02) and autoradiography. 18F-FDG-PET, DCE-MRI, and NIRF signals were also significantly higher in atherosclerotic rabbit aortas compared to controls (Fig. 2). Enhanced liver uptake was also noted in atherosclerotic animals, confirmed by the presence of MAA epitopes by immunostaining.
The human Fab antibody LA25 targeting the malondialdehyde-acetaldehyde epitope detects clinically relevant atherothrombotic lesions and allows non-invasive PET imaging of atherosclerotic plaques in rabbits. Ultimately, this radiotracer could serve as a marker to evaluate and inform therapeutic interventions.
1. Miller YI, Choi SH, Wiesner P et al. Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity. Circ Res 2011;108:235-48.
2. Binder CJ, Papac-Milicevic N and Witztum JL. Innate sensing of oxidation-specific epitopes in health and disease. Nat Rev Immunol 2016;16:485-97.
3. Dweck MR, Aikawa E, Newby DE, Tarkin JM, Rudd JH, Narula J and Fayad ZA. Noninvasive Molecular Imaging of Disease Activity in Atherosclerosis. Circ Res 2016;119:330-40.
Fondation Leducq, R01-HL119828, R01-HL078610, R01-HL106579, R01 HL128550, R01 HL136098, P01 HL136275, R35 HL135737, P01-HL055798, (ST and/or JLW), R01 EB009638 (ZAF), P01-HL131478 and R01-HL125703 (WJMM), and AHA 16SDG31390007 (CPM). MLS is supported by AHA 17PRE33660729 and the Foundation “De Drie Lichten” in The Netherlands.
Figure 1. 89Zr-LA25 evaluation in Apoe-/- mice
Blood time-activity curve (A), aortic accumulation (B-C), and radioactivity distribution in selected tissues 4 hours post injection (D) of 89Zr-LA25 or control Fab 89Zr-LA24 in Apoe-/- mice. Immunofluorescence and autoradiography on aortic sections from Apoe-/- mice after 89Zr-LA25 injection showing radioactivity deposition on macrophage-rich (CD68) areas (E).
Figure 2. Phenotyping of rabbit atherosclerotic plaques by PET/MRI.
Representative coronal aortic fused PET/MR imaging 24 hours p.i. of 89Zr-LA25 (A), autoradiographs and gamma counting (whole aortas) 28 hours p.i. of 89Zr-LA25 (B), MR T2-weighted imaging (C), 18F-FDG PET/MRI (D), DCE-MRI (E), and DiD-rHDL near-infrared fluorescence imaging (F), in healthy control (white) and atherosclerotic abdominal aortas (black).
Keywords: PET/MRI, Atherosclerosis, oxidation specific epitopes, molecular imaging
Platelet-targeted microbubbles for diagnostic and theranostic approach of thrombosis using in vivo molecular ultrasound imaigng: Treatment without bleeding complications (#325)
X. Wang1, 2, Y. Gkanatsas1, K. Peter1, 2
1 Baker Heart and Diabetes Institute, Atherothrombosis and Vascular Biology, Melbourne, VIC, Australia
Most acute cases of myocardial infarction and stroke are caused by atherothrombosis, when platelet adhesion, activation and aggregation, lead to thrombus formation and vessel occlusion. Glycoprotein (GP) IIb/IIIa complex, is the most abundant receptor expressed on the platelet surface, responsible for adhesion and aggregation. We have developed conformation-specific single-chain antibodies (scFv) that bind specifically to ligand-induced binding sites (LIBS) on activated GPIIb/IIIa.
For diagnostic imaging, MB were conjugated to either a single-chain antibody (scFv) specific for activated GPIIb/IIIa (LIBS-MB), or a non-specific scFv (control-MB). For theranostic approach, we conjugated thrombolytic drugs, such as single chain urokinase plasminogen activator (scuPA), to form targeted theranostic MBs (TT-MB). In a ferric chloride induced carotid artery thrombi mouse model, imaging was performed before and after MB injection.
LIBS-MBs strongly adhered to immobilized activated platelets and micro-thrombi under flow. A significant increase in decibel (dB) was observed after LIBS-MB but not after control-MB injection (9.55 ± 1.7 versus 1.46 ± 1.3 dB; p<0.01). For theranostic approach, TT-MB significantly reduced thrombus size after 45 min, while no significant difference was noted in the MB that were targeted but without urokinase (37 ± 6 vs. 97 ± 4, mean % change ± SEM, normalized to baseline thrombus size, p<0.001). The same degree of efficient thrombolysis was only achievable using a high dose of urokinase (NS). The targeting and thus clot-enrichment effect of TT-MBs results in a highly potent fibrinolysis that could only be matched using high doses of non-targeted urokinase. However, the latter is associated with a highly prolonged bleeding time (79 ± 7 vs. 1079 ± 261, sec ± SEM, p<0.001). In contrast, TT-MB does not prolong bleeding time (NS).
Our LIBS-MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis in vivo. TT-MBs conjugated with recombinant urokinase represent a novel and unique theranostic approach to simultaneously diagnose thrombosis, as well as to treat and monitor the success or failure of thrombolysis. This unique, non-invasive and cost effective technology holds promise for major progress towards rapid diagnosis and bleeding-free, potent therapy of the vast number of patients suffering from thrombotic diseases.
Keywords: ultrasound, platelets, thrombus, theranostics
Nanobody-facilitated multiparametric PET/MRI phenotyping of atherosclerosis (#81)
M. L. Senders1, 2, S. Hernot3, G. Carlucci4, 5, J. C. van de Voort2, F. Fay2, 6, C. Calcagno2, J. Tang2, A. Alaarg2, Y. Zhao2, S. Ishino2, A. Palmisano2, G. Boeykens2, A. E. Meerwaldt2, B. L. Sanchez-Gaytan2, S. Baxter2, L. Zendman2, M. E. Lobatto2, 7, N. A. Karakatsanis2, P. M. Robson2, A. Broisat8, G. Raes9, 10, J. S. Lewis5, 11, 12, S. Tsimikas13, T. Reiner5, 11, Z. A. Fayad2, N. Devoogdt3, W. J. M. Mulder1, 2, C. Pérez-Medina2
1 Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, United States of America
Non-invasive characterization of atherosclerosis, the pathophysiological process of stroke and myocardial infarction1, remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated2. Here, we aimed to combine target-specific nanobody-positron emission tomography (PET) imaging information with functional and anatomical magnetic resonance imaging (MRI) readouts to develop an integrative multiparametric atherosclerotic plaque phenotyping procedure.
We screened three nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule 1 (VCAM-1), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with Copper-64 (64Cu), were extensively evaluated in Apoe-/- mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography and histological analyses.
The three nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with Gallium-68 (68Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68Ga-MMR-PET, plaque burden by T2-weighted MRI (T2W-MRI), and neovascularization by dynamic contrast enhanced MRI (DCE-MRI). Additionally, inflammation and microcalcifications were evaluated by 18F-fluorodeoxyglucose and 18F-sodium fluoride PET, respectively. Using our multiparametric approach, we were able to detect an increase in macrophage and plaque burden, neovascularization, inflammation and microcalcifications as disease progressed, and correlated with histopathological features.
We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MR imaging protocol that allows non-invasive assessment of different processes relevant to atherosclerosis progression. This multimodal imaging approach would not only have a potential impact on future anti-atherosclerosis clinical drug trials, but is immediately relevant on a preclinical level, both for a better understanding of atherosclerosis biology and the development and evaluation of new drugs, noninvasively and longitudinally in animals.
1. Hansson GK. Inflammation, Atherosclerosis, and Coronary Artery Disease. N Engl J Med. 2005;352(16):1685-1695.
2. Dweck MR, Aikawa E, Newby DE, Tarkin JM, Rudd JHF, Narula J, Fayad ZA. Noninvasive Molecular Imaging of Disease Activity in Atherosclerosis. Circ Res. 2016;119(2):330-340.
This work was supported by the National Institutes of Health grants R01 EB009638, P01 HL131478 (Z.A.F.), R01 HL125703, R01 HL118440 (W.J.M.M.), P30 CA008748, the American Heart Association 16SDG31390007 (C.P.M.), 17PRE33660729 (M.L.S.), the Netherlands Organization for Scientific Research NWO Vidi (W.J.M.M) and the “De Drie Lichten” Foundation in The Netherlands (M.L.S.). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) for financial support (T.R.).
Figure 1. Nanobody-radiotracer screening in mice.
Radioactivity distribution at 3 h p.i. of 64Cu-nanobodies. B) Autoradiography (AR) and C) radioactivity concentration in aortas at 3 h p.i. D) PET/CT images 1 h p.i. of 64Cu-VCAM (left) and 64Cu-MMR (right). E) Aortic root sections showing H&E staining (top left), AR (top right), CD31 (endothelial cells, bottom left) and CD68 (macrophages, bottom right) immunostaining. All in Apoe-/- mice.
Figure 2. PET/MRI evaluation of atherosclerosis progression.
A) Coronal aortic PET/MR images for 18F-FDG (left), 68Ga-MMR (middle) and 18F-NaF (right), and B) T2W-MRI (left) and DCE-MRI (right) images from healthy and atherosclerotic rabbits (4 or 8 months on high-fat diet, HFD). C) Cardiac PET/MR images and aorta-to-heart ratios in rabbits with atherosclerosis (8HFD). D) Aortic sections from rabbits stained with H&E and RAM-11 (macrophages). * P < 0.05.
Keywords: Atherosclerosis, PET/MRI, nanobody, molecular imaging
Quantitative Intravascular Fluorescence-Ultrasound Imaging In Vivo (#58)
D. Bozhko1, V. Ntziachristos1
1 Technische Universität München, CBI, Munich, Bavaria, Germany
The need to identify and quantify the vulnerability state of the atherosclerotic plaques led to the development of multi-modality intravascular imaging systems with molecular sensitivity [1,2]. While intravascular ultrasound (IVUS) imaging and optical coherence tomography (OCT) reveal information related to arterial structures and stents, they fail to assess the biological features of vascular disease. To enable a co-registration of molecular and morphological aspects of arterial disease in vivo a hybrid near-infrared fluorescence - intravascular ultrasound (NIRF-IVUS) imaging was introduced.
Fully integrated NIRF-IVUS catheter was engineered to accurately co-register biological and morphological readings in vivo. A correction algorithm utilizing IVUS information was developed to account for the distance-related fluorescence attenuation due to through-blood imaging. NIRF-IVUS was validated in various clinically-relevant scenarios including a model of angioplasty-induced vascular injury and a model of fibrin deposition on coronary artery stents in pigs.
Co-registration of NIRF and IVUS signals, and a NIRF attenuation blood correction model, were verified to be physically accurate in vitro. We discovered that distance-correction model calibrated empirically on ex vivo data overestimates the degree of light attenuation that occurs in vivo. Therefore, we calibrated our model based on in vivo measurements. Next, concurrent in vivo intravascular imaging was performed through flowing blood. The addition of ICG-enhanced NIRF assessment improved the detection of angioplasty-induced endothelial damage compared to standalone IVUS. NIRF detection of coronary stent fibrin illuminated stent pathobiology that was concealed on standalone IVUS. Fluorescence reflectance imaging and microscopy of resected tissues corroborated the in vivo findings.
Integrated NIRF-IVUS enables simultaneous co-registered through-blood imaging of disease related morphological and biological alterations in coronary and peripheral arteries in vivo. Clinical translation of NIRF-IVUS may significantly enhance knowledge of arterial pathobiology, leading to improvements in clinical diagnosis and prognosis, and help guide the development of new therapeutic approaches for arterial diseases.
1. Bourantas C V., Jaffer FA, Gijsen FJH, Soest G van, Madden SP, Courtney BK, Fard AM, Tenekecioglu E, Zeng Y, Steen AFW van der, Emelianov S, Muller J, Stone PH, Marcu L, Tearney GJ, Serruys PW. Hybrid intravascular imaging: recent advances, technical considerations, and current applications in the study of plaque pathophysiology. Eur Heart J 2016;ehw097.
2. Ma T, Zhou B, Hsiai TK, Shung KK. A Review of Intravascular Ultrasound-based Multimodal Intravascular Imaging: The Synergistic Approach to Characterizing Vulnerable Plaques. Ultrason Imaging 2016;38:314–331.
Schematic of the NIRF-IVUS imaging system for intravascular through-blood imaging.
The NIRF-IVUS imaging catheters (insets on top left) consist of an ultrasound transducer and NIRF optical fiber. The dual-modality imaging probe rotates and pulls back inside a transparent catheter sheath using an electro-optical rotary joint, which connects the moving catheter with the stationary back-end console (insets on bottom right).
Keywords: Ultrasound, near-infrared, fluorescence, intravascular, molecular, hybrid, imaging, atherosclerosis, CAD, cardiovascular
Non-invasive imaging of macrophage-rich atherosclerotic plaques using Zirconium-89 (89Zr)-labelled desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO)-coupled anti-Galectine-3 (Gal-3)-F(ab')2. (#374)
Z. Varasteh1, F. De Rose1, S. Mohanta2, Y. Li2, M. Braeuer1, B. Miritsch3, S. Nekolla1, A. Habenicht2, H. Sager3, A. Bartolazzi4, M. Schwaiger1, C. D'Alessandria1
1 Klinikum rechts der Isar-TUM, Nuclear medicine, Munich, Bavaria, Germany
Atherosclerosis remains the main cause of mortality in industrialized countries. Identification of the lesions prone to rupture, may lead to the application of pharmacological/mechanical strategies to prevent clinical events. High macrophage density is considered as potential marker of plaque vulnerability. Galectin-3 (Gal-3) has been reported to be expressed on activated macrophages. In the present project, our aim was to evaluate the potential of 89Zr-anti-Gal-3-F(ab')2 to selectively target infiltrated macrophages and non-invasively image atherosclerotic plaques in ApoE-KO mice using PET.
DFO-anti-Gal-3-F(ab')2 was labelled with 89Zr. The binding selectivity of the fluorescent and 89Zr-labelled tracer was evaluated in vitro, on M0, M1 and M2 activated macrophages. The radiotracer was injected into ApoE-KO and age-matched control mice. Animals were scanned 48 h p.i. Upon PET/CT scans, mice were sacrificed and organs were collected for radioactivity measurement. The whole length aortas were harvested free from adipose tissue for Sudan-IV staining and autoradiography. Cryosections were prepared for immunofluorescence staining (IFS).
Fluorescent and 89Zr-labelled tracer accumulated in vitro mainly in M2 activated macrophages (Figure 1). 89Zr-DFO-anti-Gal-3-F(ab')2 accumulated in vivo in the liver (7.8±1.3 %ID/g), spleen (13.7±3.2 %ID/g) and kidneys (69±7 %ID/g, 48 h p.i.). It showed only low residual blood signal (0.5±0.1 %ID/g, 48 h p.i.). Focal signals could be detected in the atherosclerotic plaques of ApoE-KO mice (Figure 2) whereas no signal was detected in the aortas extracted from control mice. 89Zr-DFO-anti-Gal-3-F(ab')2 uptake was observed in atherosclerotic plaques on autoradiography correlating well with Sudan-IV-positive areas. The 89Zr-DFO-anti-Gal-3-F(ab')2 uptake in the plaques was associated with subendothelial accumulations of Gal-3 expressing CD68-positive macrophages confirmed by IFS. No Gal-3 expression was observed in the adventitia and adipose tissue. In the plaques, the Gal-3 expression was higher in the shoulder region (Figure 2).
Our data suggest that 89Zr-DFO-anti-Gal-3-F(ab')2 may serve as both an imaging agent for macrophage-rich plaques and a novel therapy assessment tracer in anti-inflammatory strategies for the treatment of atherosclerosis. Small dimension atherosclerotic plaques could be efficiently targeted and visualized in vivo using 89Zr-DFO-anti-Gal-3-F(ab')2 in ApoE-KO mouse model. However, substantial uptake of 89Zr-DFO-anti-Gal-3-F(ab')2 in the liver, spleen and the kidneys may impede the interpretation of the signals originating from abdominal aorta in mice.
In vivo PET/CT and ex vivo IFS
Axial, coronal and sagittal views of PET/CT images acquired in vivo 48 h p.i. of 89Zr-DFO-anti-Gal-3-F(ab')2. Note the intense focal signal in the atherosclerotic plaque of the aortic arch in an ApoE-KO mouse (white arrows). Photomicrographs of Gal-3 (green), CD68 (red) immunofluorescence staining. Overlaping domains of expression is shown in yellow. DAPI stained nuclei are shown in blue.
Keywords: Atherosclerotic plaques, Galectin-3, Non-invasive imaging