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8-COLOUR MULTIPLEX IMMUNOHISTOCHEMISTRY FOR DEEP PHENOTYPING OF THE IMMUNOLOGICAL RESPONSE IN HUMAN AORTOPATHIES. (#535)
A. Staal1, M. Gorris1, I. Wortel1, A. Duffelen1, G. Geuzebroek2, J. Textor1, B. Kusters4, W. Morshuis2, J. de Vries1, J. Timmermans3, R. van Kimmenade3, M. Srinivas1
1 Radboudumc, Tumor Immunology Lab, Nijmegen, Netherlands
Thoracic ascending aortic aneurysms (TAAA) are a silent killer; 1/5th of patients with a TAAA will die from it, often before being diagnosed. TAAAs are at constant risk of acutely progressing towards an aortic dissection, necessitating immediate high risk aortic replacement, associated with very high mortality. Timely discovery and decision making is greatly hampered by insufficient knowledge on TAAA disease pathophysiology. In situ phenotyping of cells to gain insight into pathophysiological mechanisms, and to enable development of new diagnostic, prognostic, and medical strategies.
TAAA and Stanford type-A aortic dissection (AD) tissue samples and clinical data were collected from patients with a preventive ascending aortic replacement or acute AD respectively. To identify cells of the immune system within tissue samples, two 8-color multiplex panels staining relevant players of the adaptive and innate immune system on a single slice per panel, were developed. Multispectral images of the samples were acquired with the Vectra microscopy system and were subsequently unmixed and processed with inForm software. With in-house developed processing software, cells can be quantified. Additionally, since tissue integrity is maintained with this technique, spatial analysis, including localization, penetration, clustering, and nearest-neighbour analysis, is possible.
With the adaptive immune cell panel, B and T-lymphocytes, and their interaction with dendritic cells, could be identified. The panel designed to classify the innate immune cells could assess MMP9 production by cells and macrophages polarisation (M1/M2). Preliminary data indicates the accumulation of immune cells, polarised towards and inflammatory phenotype, in dissected specimens.
Here we show a powerful immunohistochemistry (IHC) method, capable of deep phenotyping a large number of cells numbers, while preserving the integrity of the original tissue. Detection of specific immune infiltrates could lead to a hypothesis-driven diagnostic marker for aortic disorders.
Keywords: Aortic Dissection, Immunohistochemistry, aortic aneurysm
Assessment of Cardiac Morphology and Function in Experimental Pulmonary Arterial Hypertension by MicroCT (#561)
F. Ruscitti1, S. Belenkov2, S. Cavalli1, F. Pastore1, B. Kojonazarov3, F. F. Stellari1, R. Schermuly3, G. Villetti1, F. Facchinetti1, M. Civelli1, S. Cantoni1
1 Chiesi S.p.A, Pre-Clinical R&D, Parma, Italy
Micro-computed tomography (MicroCT) is a non-invasive imaging technique that provides quantitative morphologic and functional data non-destructively and longitudinally. The goal of this study was to evaluate the value of MicroCT in the Sugen 5416 chronic hypoxia rat model of Pulmonary Arterial Hypertension (PAH) and to compare hemodynamics with volume-based indices measured by MicroCT.
PAH was induced in male SD rats by a single subcutaneous injection of Sugen 5416 (20 mg/kg) followed by 3 weeks of hypoxia (10% oxygen) with subsequent normoxic conditions for 3 (group H_A) and 2 (group H_B) weeks. Left and right ventricle (LV, RV), end-systolic and end-diastolic volumes (ESV and EDV), stroke volumes (SV), % of ejection fraction (EF%) and mass were derived from MicroCT Quantum GX (PerkinElmer, Inc.) measurements in conjunction with eXIA160XL blood-pool contrast agent. Systolic right ventricular pressure (RVP) and systemic arterial pressure (SAP) were measured before animal sacrifice by using a Millar catheter (SPR320, 2F), and RV hypertrophy was assessed post-mortem by measuring mass ratio of RV and left ventricle plus interventricular septum (LV+S).
Volume-based indices measured by MicroCT indicated clear signs of RVH and RV systolic dysfunction in PAH rats compared with control animals. The alterations were less pronounced in rats from group B (2 weeks of normoxia) than in rats from group A (3 weeks of normoxia). A good linear correlation was observed between the microCT-derived indices, hemodynamics, and post-mortem myocardial mass measurements (Fig. 1).
MicroCT-derived volume-based indices can accurately reflect both LV and RV morphology and function, and could be used as predictors for longitudinal monitoring of disease progression and treatment efficacy in rodent models of PAH.
Keywords: Pulmonary Hypertension, Micro-CT, imaging, Sugen-Hypoxia, heart failure
Impact of empagliflozin on cardiac energy status and function in diabetic db/db mice (#487)
D. Abdurrachim1, E. Manders1, K. Nicolay1, E. Mayoux2, J. J. Prompers1, 3
1 Eindhoven University of Technology, Biomedical Engineering, Eindhoven, Netherlands
Diabetes is associated with impaired cardiac energetics and diastolic dysfunction.1,2 In the EMPA-REG OUTCOME trial, the sodium glucose cotransporter 2 inhibitor empagliflozin showed impressive benefits on cardiovascular outcome in type 2 diabetes patients.3 A shift in myocardial fuel metabolism toward utilization of ketones, a more energy-efficient fuel, has been proposed to explain these benefits.4 Our aim was to investigate the effects of empagliflozin on in vivo cardiac energy status and function in diabetic db/db mice using 31P MRS and MRI, respectively.
Male diabetic db/db mice were randomized into a placebo group (N=10; placebo) or an empagliflozin group (N=11; EMPA) to study the effects of acute (single dose) and chronic (6 weeks) treatment. On day 1, animals were fasted for 4 hours after which they received a single dose of empagliflozin (30 mg/kg body weight) in 0.5% Natrosol (EMPA) or 0.5% Natrosol (placebo) by oral gavage. After another 2 hours of fasting, plasma glucose and ketone levels were measured. Thereafter, cine MRI and 31P MRS were performed at a 9.4 T MR scanner (Bruker). For the EMPA group, treatment was continued for another 6 weeks by mixing empagliflozin into the chow (resulting in an average dose of 45 ± 6 mg per kg body weight per day). After 6 weeks of treatment, 31P MRS and cine MRI measurements were repeated.
Plasma glucose levels were lower in EMPA compared with placebo, both after a single dose of empagliflozin and after 6 weeks of treatment. After a single dose, plasma ketone levels were higher in EMPA compared with placebo, but after 6 weeks ketone levels were not different between groups.
After a single dose of empagliflozin, the cardiac PCr/ATP ratio was 44% higher in EMPA than in placebo (Figure 1). However, after 6 weeks of treatment, the PCr/ATP ratio in EMPA was no longer different from placebo.
End-diastolic volume (EDV) and stroke volume (SV) were lower compared with placebo after a single dose of empagliflozin, but these differences disappeared after 6 weeks of treatment (Figure 2). Ejection fraction and cardiac output were not significantly different between groups. After 6 weeks of treatment, the ratio of early to late peak filling rates (E/A ratio) tended to be higher in EMPA compared with placebo (Figure 2).
Empagliflozin acutely improved cardiac energy status in a mouse model of diabetes, which was correlated with higher plasma ketone levels and reduced end-diastolic and stroke volumes, indicating possible roles for empagliflozin in modulating cardiac fuel use4 and/or cardiac pre- and after-load. However, while cardiac diastolic function tended to be improved after 6 weeks of treatment with empagliflozin, cardiac PCr/ATP ratio, end-diastolic and stroke volumes, and plasma ketone levels were similar to placebo.
1. Diamant M, Lamb HJ, Groeneveld Y, Endert EL, Smit JW, Bax JJ, Romijn JA, de Roos A, Radder JK. Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus. J Am Coll Cardiol. 2003;42:328-335
2. Scheuermann-Freestone M, Madsen PL, Manners D, Blamire AM, Buckingham RE, Styles P, Radda GK, Neubauer S, Clarke K. Abnormal cardiac and skeletal muscle energy metabolism in patients with type 2 diabetes. Circulation. 2003;107:3040-3046
3. Zinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, Mattheus M, Devins T, Johansen OE, Woerle HJ, Broedl UC, Inzucchi SE. Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes. New England Journal of Medicine. 2015;373:2117-2128
4. Ferrannini E, Mark M, Mayoux E. CV protection in the EMPA-REG OUTCOME trial: A "thrifty substrate" hypothesis. Diabetes Care. 2016;39:1108-1114
Figure 1. Cardiac energetics as measured by 31P MRS.
(A) Representative cardiac MR images, with the white rectangles indicating the localization of the voxel for 31P MRS. (B) Representative cardiac 31P MR spectra from a single-dose placebo-treated and empagliflozin-treated mouse. (C) Quantification of PCr/γ-ATP ratio. *P<0.05 vs. placebo at the same time point, #P<0.05 vs. single dose for the same group.
Figure 2. Cardiac function as measured by MRI.
Quantification of left-ventricular (A) end-diastolic volume, (B) end-systolic volume, (C) stroke volume, (D) ejection fraction, (E) cardiac output, and (F) E/A ratio. *P<0.05 vs. placebo at the same time point, #P<0.05 vs. single dose for the same group, ‡P<0.05 vs. single dose independent of group.
Keywords: Magnetic resonance imaging, Cardiac metabolism, Cardiac energetics, Magnetic resonance spectrsocopy, Cardiac function
Regional kinetic modelling of 18F-FDG PET in combination with non-invasive MRI; an improved method for tissue characterisation in a rat model of myocardial infarction (#363)
M. A. Jansen1, L. Le Page1, J. Kwiecinski1, R. J. Lennen1, A. Thomson1, G. A. Gray1, A. A. Tavares1
1 University of Edinburgh, Centre for Cardiovascular Science, Edinburgh, United Kingdom
Biomarkers to predict and rank the success of acute myocardial infarction (MI) therapies are in increasing demand, as new therapies targeting the fine balance of inflammation and tissue repair emerge. Non-invasive imaging biomarkers can be pivotal to this process. 18F-FDG positron emission tomography (PET) and magnetic resonance imaging (MRI) are widely used for clinical routine imaging. This work aimed to develop an improved approach to characterise the myocardial response post MI based on kinetic modelling of 18F-FDG-PET data in combination with typical MRI data.
Sprague-Dawley rats were split into three groups. Sham and MI animals underwent surgery; the left anterior descending coronary artery was occluded for 30 min in the MI group to induce an infarct. Sham animals were opened with no occlusion; naïve animals underwent no surgery. Animals were scanned using MRI (to assess infarct size and cardiac function) and dynamic 18F-FDG-PET at 48h post-surgery; a subset of animals was also scanned at 3 weeks post MI. Kinetic modelling of acquired PET data was performed using the Patlak plot and compared to standard uptake values (SUVs). Hearts from animals culled 48h post-surgery were stained using triphenyl tetrazolium chloride (TTC) to demonstrate presence of infarct ex vivo.
MRI and TTC staining confirmed the presence of infarcts in the MI animals (Figure 1). Regional kinetic modelling of the 18F-FDG-PET data demonstrated a relative increase in glucose uptake in the infarct area early during infarct healing, which was decreased by 3 weeks, when infarct tissue was replaced by scar. Also observed was a decrease in glucose uptake in the septal wall region in both the sham and MI animals at 48h post-surgery, which was no longer present at 3 weeks. We created a reference database for small animal 18F-FDG-PET measurements in healthy naïve, sham and myocardial infarct rats based on the American Heart Association 17 segment model. An example of this is shown in Figure 2 for hearts assessed 48h after MI, where the numbers indicate the percentage change of the metabolic rate of glucose uptake for each segment relative to that of total myocardium.
The use of kinetic analysis to interpret 18F-FDG-PET data can provide valuable, specific information on infarct repair post-MI, both during the early inflammatory phase and following replacement of necrotic tissue by scar tissue; these data provide more objective information than is obtained using SUV analysis. Further, the reference database created in this study can be easily translated to clinical use and may assist with prediction and ranking of ongoing or novel MI therapies.
Keywords: FDG-PET, MRI, Myocardial infarction, rat model
Radiolabelling of Calcium Phosphates Nanoparticles for assessing their ability in cardiac targeting: In vitro Stability and Preliminary In vivo Evaluation with Scintigraphic/X-ray imaging (#64)
E. Fragogeorgi1, S. Sarpaki2, M. Rouchota2, A. Adamiano3, M. Iafisco4, M. Georgiou2, P. Bouziotis1, D. Catalucci4, G. Loudos2, 5
1 National Center for Scientific Research (NCSR) “Demokritos”, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, Athens, Greece
Synthetic Calcium Phosphate Nanoparticles’ (CaP-NPs) excellent properties (biocompatibility, non-toxicity, etc.) renders them excellent candidates as potential multimodal imaging agents and drug carriers.[1-3] In the frame of the CUPIDO project, we radiolabeled CaP-NPs and we investigated their in vitro stability and in vivo profile in normal mice through biodistribution and scintigraphic/x-ray studies. Results of intravenous administration route are presented. Having the targeting of the heart through the lungs as the ultimate goal, data from inhalation are currently under development.
For radiolabeling of CaP-N, the metastable isotope of technetium (99mTc) was used via the preparation of 99mTc-MDP conjugate. Aliquots of 99mTc-MDP were added to CaP-NPs suspension and centrifuged. The radioactivity in the pellet and the supernatant were measured to provide the percentage of bound radioactivity. Quality control of 99mTc-MDP-CaP-NPs resuspended pellet was carried out with paper chromatography. An in vitro stability assay was performed from t=0 up to 24h post-preparation, over a range of temperatures (5oC, 25oC, 37oC) and under different incubation conditions in both aqueous solution (saline 0.9% v/v and glucose (5%)) and human serum at 37oC. Ex vivo and scintigraphic/x-ray fused imaging profile in normal mice was studied by intravenously injecting CaP-NPs in glucose (5%).
The radiochemical yield for 99mTc-MDP-Cap-NPs was 81.5 ± 8.5 % providing a single radioactive species (RCP:> 98 %). In vitro stability up to 24 h post-labelling was high (>98 %) at all different sets of temperature and while diluted ten times in aqueous conditions [saline: 80-90 % and glucose 5 %: 90-95 % and in biological media (human serum: >92 %]. Radioactivity was mainly found in liver, spleen and lungs up to 2 h post injection (p.i.) and in liver and spleen at 24 h p.i. based on biodistribution results and was confirmed by imaging results.
The encouraging results, allow the use of CaP-NP as a suitable approach for other routes of administration like gavage or inhalation. Our data are in good agreement with published work on intravenously injected hydroxyapatite nanoparticles.[2,3]
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 720834 and from the program of Industrial Scholarships of Stavros Niarchos Foundation.
Keywords: calcium phosphates, scintigraphic/x-ray imaging, nanoparticles, administration route
Evaluation of 68Gallium-labeled peptide for the detection of metalloproteinase 2/9 expression in mouse atherosclerotic plaques (#293)
M. Kiugel1, S. Hellberg1, M. Käkelä1, H. Liljenbäck1, 2, T. Saanijoki1, J. Tuomela3, J. Knuuti1, 4, A. Saraste1, 4, 5, A. Roivainen1, 2, 4
1 University of Turku, Turku PET Centre, Turku, Finland
Expression of matrix metalloproteinases 2/9 (MMP-2/9) has been implicated in arterial remodeling and inflammation in atherosclerosis. We evaluated a 68Ga-labeled peptide for the detection of MMP-2/9 in atherosclerotic mouse aorta.
We studied nine low-density lipoprotein receptor deficient mice (LDLR-/-ApoB100/100) kept on a Western-type diet. Distribution of intravenously injected MMP-2/9-targeting 68Ga-DOTA-peptide was studied by combined positron emission tomography (PET) and contrast-enhanced computed tomography (CT). At 60 min post-injection, aortas were cut into cryosections for autoradiography analysis of tracer uptake, histology, and immunohistochemistry. Zymography was used to assess MMP-2/9 activation and pre-treatment with MMP-2/9 inhibitor to assess the specificity of tracer uptake.
Tracer uptake was not visible by in vivo PET/CT in the atherosclerotic aorta, but ex vivo autoradiography revealed 1.8 ± 0.34 times higher tracer uptake in atherosclerotic plaques than in normal vessel wall (p = 0.0029). Tracer uptake in plaques correlated strongly with the quantity of Mac-3-positive macrophages (R = 0.91, p < 0.001), but weakly with MMP-9 staining (R = 0.40, p = 0.099). Zymography showed MMP-2 activation in the aorta, and pre-treatment with MMP-2/9 inhibitor decreased tracer uptake by 55% (p = 0.0020).
The MMP-2/9-targeting 68Ga-DOTA-peptide shows specific uptake in inflamed atherosclerotic lesions; however, a low target-to-background ratio precluded in vivo vascular imaging.
The studies were conducted within the Finnish Centre of Excellence in Molecular Imaging in Cardiovascular and Metabolic Disease supported by the Academy of Finland, the University of Turku, the Turku University Hospital, and Åbo Akademi University. This study was also funded by the Hospital District of Southwest Finland/Turku University Hospital (ERVA #13856, #13119, and #13260), Academy of Finland (#258814), Sigrid Jusélius Foundation, the Finnish Foundation for Cardiovascular Research, the Maud Kuistila Memorial Foundation, Ida Montin’s foundation, Emil Aaltonen foundation, and the Finnish Cultural Foundation.
Distribution of (68)Ga-DOTA-peptide in atherosclerotic mouse aorta
Digital autoradiography (a) compared to H&E staining (b). c: average tracer accumulation in the adventitia, normal vessel wall, and atherosclerotic plaques. Adjacent sections are stained with Movat (d), MMP-9- (e), or Mac-3 antibody (detecting macrophages) (f). A, adventitia; B, brachiocephalic artery; L, lumen; LC, left carotid artery; LS, left subclavian artery; P, plaque; W, wall.
Correlations between tracer uptake and immunohistology
Scatter plots show correlations between areal percentages of Mac-3-positive macrophages (a), or MMP-9 (b), and corresponding 68Ga-DOTA-peptide uptake in the atherosclerotic plaques. Each symbol type represents plaques from the same animal. R, Pearson’s rank correlation coefficient; PSL, photostimulated luminescence.
Improved detection of molecular markers of atherosclerotic plaques using sub-millimetre PET imaging (#427)
J. Bridoux1, S. Neyt2, P. Debie1, B. Descamps3, N. Devoogdt1, F. Cleeren4, G. Bormans4, A. Broisat5, C. Xavier1, C. Vanhove3, S. Hernot1
1 Vrije Universiteit Brussel, Laboratory of In vivo Cellular and Molecular Imaging, ICMI-BEFY, Brussels, Belgium
PET is the preferred modality to detect atherosclerotic plaque inflammation because it is the most sensitive and quantitative molecular imaging modality. For plaque imaging, sensitivity and resolution are particularly important parameters as lesions are small and sparse.
The aim of this study was to evaluate a novel 18F-analogue of the previously developed VCAM-1-specific Nanobody (Nb)1 as tracer for atherosclerosis, and investigate whether the use of a PET system with sub-millimetre spatial resolution and increased sensitivity, can further improve detectability.
Nb cAbVCAM1-5 was radiolabeled with 18F via the Al18F-labeling strategy involving the RESCA chelator ([18F]AlF(RESCA)-Nb). ApoE-/- mice (n=6/group) were injected intravenously with ([18F]AlF(RESCA)-Nb (14±9MBq). ApoE-/- co-injected with excess of unlabelled Nb (14±8MBq) served as a control group. At 2.5h p.i., mice were imaged sequentially using a cross-over design on two different PET/CT systems: a LabPET8 (TriFoil Imaging, Chatsworth, USA) and a b-CUBE (MOLECUBES, Ghent, Belgium) with 1.2 and 0.8mm spatial resolution, respectively. Volumes-of-interest were drawn at the level of the aortic arch, brain and heart, and target-to-brain (T/B) and target-to-heart (T/H) ratios were calculated. At the end, animals were killed for ex vivo biodistribution and autoradiography of the excised aorta.
cAbVCAM1-5, randomly conjugated with RESCA, was successfully radiolabelled with Al18F (radiochemical yield 78±2% and radiochemical purity >99%). [18F]AlF(RESCA)-Nb showed specific accumulation in atherosclerotic lesions (1.64±0.36 times higher than control group), as could be visualized using both PET/CT imaging systems (Figure 1) and confirmed by ex vivo analysis and autoradiography. Better image quality was nevertheless achieved with the b-CUBE. Indeed, significantly higher T/H and T/B ratios were obtained with the b-CUBE than with the LabPET8 (T/H: 1.75±0.30 vs 1.40±0.24, p<0.05; T/B: 3.88±0.88 vs 2.57±0.54, p<0.05). Besides uptake of the Nb-tracer in expected organs and tissues (kidneys, bladder and spleen), substantial accumulation of [18F]AlF(RESCA)-Nb (or degradation products thereof) was observed in bone structures. This uptake was not considered specific targeting as it could not be reduced by competition.
[18F]AlF(RESCA)-labeled anti-VCAM1 Nb enables PET/CT imaging of atherosclerotic plaque inflammation, although non-specific bone uptake related to the radiolabeling strategy is undesirable for low expression targets. Furthermore, we demonstrated that using the latest PET technology, offering sub-mm spatial resolution, improved detection of molecular markers of atherosclerotic plaques can be obtained.
This project was funded by the Flanders Research Foundation (FWO) (G005815N)
Keywords: nanobodies, atherosclerosis, PET imaging, 18F-AlF
Optoacoustic and ultrasound biomicroscopy of skull microvasculature in mice (#508)
H. Estrada1, J. Rebling1, 2, U. Hofmann1, S. Gottschalk1, D. Razansky1, 2
1 Helmholtz Center Munich, Institute for Biological and Medical Imaging, Neuherberg, Bavaria, Germany
Bone microvasculature plays an important role in key biological processes as a link between bone marrow, responsible for producing the all blood cells, and rest of the body . Yet, nondestructive in vivo observations of those processes with high spatial resolution over large fields of view are challenging due to the physical limitations imposed by the bone [2-4].
Here we demonstrate in vivo imaging of the skull microvasculature using an optical resolution optoacoustic microscope  integrated and coregistered with ultrasound biomicroscopy . The experiments were performed after removing the scalp of a 12 week old mouse with its intact skull rapidly imaged in the hybrid mode. A fast continuous scanning protocol was further implemented ensuring that a single 3D scan covering the entire mouse skull can be achieved in under 90 seconds.
Fig. 1 shows the intricate 3D patterns of the skull microvasculature acquired in the optical-resolution optoacoustic imaging mode at 578 nm excitation wavelength, achieving lateral resolution of 15 µm. The simultaneously acquired pulse-echo ultrasound information further allowed accurate differentiation of the skull microvasculature from the underlying brain cortical vasculature located below the skull bone.
Existing intravital fluorescence imaging and microscopy methods require specialized contrast agents and generally not suitable for large-scale imaging of skull microvasculature in rodents due to the skull curvature. Here we demonstrate the capabilities of hybrid optoacoustic-ultrasound biomicroscopy for high-resolution visualization of the skull microvasculature in vivo. Our label-free optoacoustic microscopy method uses hemoglobin as endogenous contrast and acquires 3D images of both parietal bones in a single scan, thus holding promise to elucitade biological processes now hidden inside bone
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 X. Cao, et al. (2011). “Irradiation induces bone injury by damaging bone marrow microenvironment for stem cells,” PNAS 108, 1609-1614.
 A. Köhler, et al. (2009) “Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones,” Blood 114, 290-298.
 F. Lassailly, et al. (2013). “Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells,” Blood 122, 1730-1740.
 H. Estrada, et al. (2014). “Real-time optoacoustic brain microscopy with hybrid optical and acoustic resolution,” Laser Phys. Lett. 11, 045601.
 H. Estrada, et al. (2014). “Hybrid optoacoustic and ultrasound biomicroscopy monitors laser-induced tissue modifications and magnetite nanoparticle impregnation,” Laser Phys. Lett. 11, 125601
This project received funding from the European Research Council under grant agreement ERC-2015-CoG-682379 (D.R).
Keywords: Microvasculature, Bone, Optoacoustic microscopy, Ultrasound biomicroscopy, Skull
Preclinical development of copper-64 radiolabeled lipid nanoparticles for targeted delivery in atherosclerotic plaques (#168)
J. Vigne1, 2, 6, R. Aid2, 6, G. Even2, M. Escudé3, 4, N. Anizan2, 6, P. Oliva5, V. Mourier3, 4, A. Cordaro5, F. Hyafil1, 2, 6, C. Chauvierre2, F. Rouzet1, 2, 6, D. Letourneur2, D. Le Guludec1, 2, 6, C. Cabella5, I. Texier3, 4
1 Bichat University Hospital, Nuclear medicine department, Paris, France
Atherotrombosis is one of the world leading cause of death globally . Recent results have shown accumulation of Lipidots™, 50 nm diameter nanoparticles composed of a lipid core stabilized by a biocompatible surfactant shell, in structures containing a high content of lipids . Thus the Lipidots™ shell was modified to enable the chelation of positron emitter radiometals such as Copper-64 to obtain PET probes. The proof-of-concept of radiolabeled Lipidots™ to achieve targeted delivery purposes of atherosclerotic plaques in a preclinical model of atherosclerosis was demonstrated.
After synthesis, introducing additional amphiphilic DOTA chelates, nanoparticles hydrodynamic diameter and polydispersity index (PDI) were measured using dynamic light scattering. Radiolabeling consisted in adding 200 MBq of 64CuCl2 to a mix of nanoparticles in ammonium acetate 0.1 M pH 7 and heating at 60°C during 25 min. Radiochemical purity was assessed by thin layer chromatography (TLC). ApoE KO mice aged over 35 weeks were used as preclinical model of atherosclerosis and injected with 20 MBq of Lipidots™. Control groups consisted in injecting Lipidots™ to WT mice and in injecting the same mixture except without particles in ApoE KO mice. PET/MRI acquisitions were realized 24 h after injection then mice were sacrificed. Autoradiography and Oil Red O staining were performed on aortas.
Functionalized Lipidots™ hydrodynamic diameter was 50.0 ± 0.9 (mean ± SD) nm and PDI was 0.084 ± 0.01. Radiochemical purity assessed by TLC was over 99% and pH was between 6 and 7 on final product 1 hour post-radiolabeling and filtration. PET/MRI acquisitions revealed a predominant liver and spleen uptake of 64Cu-Lipidots™, blood pool signal was still detected 24 hours post-injection. Autoradiography performed on aortas dissected from ApoE KO mice injected with particles exhibited different focal uptake that colocalized perfectly with the lipid mapping realized with Oil Red O staining. This pattern was not observed in control groups. Whole aortas standardized signal intensities from autoradiography were 8.6 ± 0.39 (n=4) for ApoE mice KO Lipidots™ group, 4.0 ± 0.96 for ApoE mice without Lipidots™ group (n= 2; p < 0.05) and 1.8 ± 0.03 for WT Lipidots™ group (n=3; p < 0.001).
Lipidots™ were efficiently radiolabeled by 64Cu to yield PET contrast agents with appropriate diameter and PDI, and seemed to significantly accumulate in atherosclerotic plaques in ApoE KO mice. Further studies will be performed using dye-loaded and 64Cu labelled nanoparticles to validate the suitability of these nanosystems in conditions where atherosclerotic plaques are involved. These nanoparticles could constitute an interesting tool for targeted delivery and imaging purposes in atherosclerosis.
1. WHO | Cardiovascular diseases (CVDs) [Internet]. WHO. [cited 2017]. Available from: http://www.who.int/mediacentre/factsheets/fs317/en/
2. J. Mérian, R. Boisgard, X. Decleves, B. Thezé, I. Texier, B. Tavitian, J. Nucl. Med. 2013, 54, 1996-2003
This work was supported by the EU (“NanoAthero” project FP7-NMP-2012-LARGE-6-309820). CEA-LETI/DTBS is part of the Arcane Labex program, funded by the French National Research Agency (ARCANE project n° ANR-12-LABX-003).
Authors thank the Accelerator for Research in Radiochemistry and Oncology at Nantes Atlantic (ARRONAX, Nantes, France) for Copper-64 supply.
Scheme and transmission electron microscopy of the Lipidots™ nanoparticles.
Left : Scheme of Lipidots™, 50 nm diameter nanoparticles composed of a lipid core stabilized by a biocompatible surfactant shell. Lipidots™ were formulated as previously described , introducing additional amphiphilic DOTA chelates (0.6% of dried formulation) in the nanoparticle shell.
Right : Transmission electron microscopy of Lipidots™.
Autoradiography and Oil Red O staining of mice aortas.
A, B, C and D left, autoradiography after 20h exposure of aortas lied in "en face" position.
A : WT mouse aorta from the control group receiving 64Cu radiolabeled Lipidots.
B : ApoE KO mouse aorta from the control group receiving the same mixture except particles.
C and D left : ApoE KO mice receiving 64Cu radiolabeled Lipidots.
D right : Oil red O staining of the same aorta represented in D left.
Keywords: Nanosystem, PET probe, Atherosclerosis, Targeted delivery
3D mapping of cardiac contraction on isolated rat heart using ultrasound ultrafast imaging (#248)
V. Finel1, P. Mateo1, C. Papadacci1, J. Provost1, M. Tanter1, M. Pernot1
1 Institut Langevin, ESPCI ParisTech, CNRS UMR 7587, INSERM U979, Université Paris 7 Paris, paris, France
Imaging the motion of the myocardium at high temporal resolution (<1ms) during cardiac contraction is crucial to analyse the myocardial activation sequence and improve the understanding of the heart’s contraction, particularly in conduction abnormalities. Electromechanical Wave Imaging (EWI)  was recently developed to map the transient motion of the myocardium following electrical activation on a 2D imaging plane. In this study, we demonstrate the feasibility of performing 3D ultrafast imaging  in isolated rat hearts within a single heart beat using a 3D ultrafast ultrasound scanner.
Imaging of three isolated rat hearts in the apical view were performed during sinus rhythm and during pacing.
A 2D matrix array (8MHz, 32x32 elements, 0.3mm pitch) driven by a customized, programmable, 1024-channel ultrasound scanner was used to perform 600-ms-long 2D plane-wave acquisitions at 3000 volumes/s.
Inter-frame axial displacements and strains were mapped in the myocardium using the Kasai algorithm and a least-squares estimator with a kernel size of 0.94mm, respectively. A correlation algorithm was then used to compute the activation times of the heart. The temperature of the heart was varied between 37 and 26°C to investigate the effects of hypothermia on cardiac activation. Two-lead electrocardiogram was recorded simultaneously during ultrasound acquisitions.
The sequence of the transient mechanical activation was imaged in 3D in the rat hearts. In sinusal rhythm, activation times were quantified and ranged between 100 ms and 120 ms after P-wave. The conduction pattern was completely modified during pacing: early mechanical activation was found at the pacing location and the activation patterns correlated with the electrical activation sequence. Hypothermia induced a reversible delay of activation patterns of the hearts: activation times of the hearts was shown to double from 37°C to 26°C.
These results show the feasibility of mapping in 3D the mechanical myocardial activation sequence within a single heart beat. The mechanical activation sequence of the myocardium was found in good agreement with the electrical activation pattern during pacing. Moreover, alteration of the activation sequence correlated strongly with the change of electrical conduction when varying the temperature. This technique may become a new tool to detect and follow abnormalities of electrical conduction in the heart in entire volumes.
 Konofagou et al., Ultrasonics, 50(2):208-15, 2010
 Provost et al., Proc. Nat. Acad. Sci., 108(21):8565-70, 2011
Keywords: cardiac activation, ultrafast ultrasound imaging, 3D imaging
Multimodal characterisation of atherosclerosis; role of enzymatic activity and nanomicelles protein corona (#243)
A. V. Lechuga-Vieco1, 2, H. Groult1, 2, J. Pellico1, 2, J. Ruiz-Cabello3, 2, F. Herranz1, 2
1 Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
Atherosclerosis detection by molecular imaging is usually performed by macrophage-mediated accumulation. However, we believe an active targeting could expand our knowledge of plaque development. Recently, we have taken this approach with microcalcifications1,2 and oxidised phospholipids.3 Here, we have applied a new type of nanomicelles of iron oxide coated with phosphatidylcholine. 4 We have used protein corona composition and the overexpression of Phosphatidylcholine-specific phospholipase-C to develop a new set of probes for atherosclerosis characterisation.5
Phosphatidylcholine-coated Nanomicelles (PC-NM) were produced in a single nanoemulsion step and used as the reference compound for MRI, biodistribution and histology. Dye-loaded PC-NM were also produced for immunohistochemistry analysis. Finally, 89Zr-labelled PC-NM were also synthesised for further characterisation of the nanoparticles accumulation in the plaque. For enzymatic activity, PC-NM were incubated and then studied by several techniques like, DLS, relaxometry and ICP. Animal experiments were performed in C57BL/6 (41 weeks old) as control mice and ApoE-/- (43 weeks old, 22 weeks on a HFD). After injection of PC-NM, atherosclerosis plaques were characterised by in vivo MRI, ex vivo fluorescence imaging, gamma counter biodistribution, immunohistochemistry and histology.
First, we studied the effect of the PC-PLC enzyme on the PC-NM by an increase in the hydrodynamic size (Fig.), an increase in the T2 relaxation times and by quantifying the amount of Phosphorous in the PC-NM. In vivo MRI clearly show the reduction of the signal in the plaque at the aortic arch (Fig.). Ex vivo fluorescence images of the aortas confirm that accumulation, together with signal in the renal bifurcation of ApoE-/- mice but not in C57BL/6 mice (Fig.). Gammacounter confirmed this biodistribution, with a significant accumulation in the aorta. We also found a much higher uptake by M1 macrophages than by M2 macrophages, suggesting a therapeutic approach. Finally, we studied the colocalisation in the plaque of the PC-NM with PC-PLC enzyme, ApoB-100 protein and macrophages. Briefly, we could calculate Pearson’s coefficient showing a quite strong colocalisation with ApoB-100, a bit lower with PC-PLC and no significant colocalisation with macrophages.
Our results show how plaque detection and characterisation can be achieved by an active approach with iron oxide nanoparticles. Furthermore, we show how protein corona can be used to our benefit for the active targeting of the disease. The selective accumulation of a new responsive nanomaterial in atherosclerotic lesions, and its detection by multimodal imaging, reinforce the possible use of PC-NM as a probe for characterising cellular traffic in the atherosclerotic plaque and opens up the possibility of new treatment options.
(1) Pellico, J.; Lechuga-Vieco, A. V.; Benito, M.; García-Segura, J. M.; Fuster, V.; Ruiz-Cabello, J.; Herranz, F.; Gar Ia-Segura, J. M.; Fuster, V.; Ruiz-Cabello, J.; Herranz, F. Microwave-Driven Synthesis of Bisphosphonate Nanoparticles Allows in Vivo Visualisation of Atherosclerotic Plaque. RSC Adv. 2015, 5 (3), 1661–1665.
(2) Salinas, B.; Ruiz-Cabello, J.; Lechuga-Vieco, A. V.; Benito, M.; Herranz, F. Surface-Functionalized Nanoparticles by Olefin Metathesis: A Chemoselective Approach for In Vivo Characterization of Atherosclerosis Plaque. Chem. - A Eur. J. 2015, 21 (29), 10450–10456.
(3) Pellico, J.; Fernandez-barahona, I.; Benito, M.; Gaitán-Simón, Á.; Gutiérrez, L.; Ruiz-Cabello, J.; Herranz, F. Hybrid Pretargeted Molecular Imaging of Atherosclerosis with Bioorthogonal Nano-Radiotracers. Bioconjug. Chem. 2017, under review.
(4) Groult, H.; Ruiz-Cabello, J.; Lechuga-Vieco, A. V.; Mateo, J.; Benito, M.; Bilbao, I.; Martínez-Alcázar, M. P.; Lopez, J. A.; Vázquez, J.; Herranz, F. F. Phosphatidylcholine-Coated Iron Oxide Nanomicelles for In Vivo Prolonged Circulation Time with an Antibiofouling Protein Corona. Chem. - A Eur. J. 2014, 20 (50), 16662–16671.
(5) Lechuga-Vieco, A.; Groult, H.; Pellico, J.; Mateo, J.; Enriquez, J. A.; Ruiz-Cabello, J.; Herranz, F. Protein Corona and Phospholipase Activity Drive Selective Accumulation of Nanomicelles in Atherosclerotic Plaques. Nanomedicine: Nanotechnology, Biology and Medicine 2017, under review.
This work was funded by the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and Carlos III Health Institute (grant number: DTS16/00059). The CNIC is supported by the MEyC and the Pro-CNIC Foundation and is a SO-Center of Excellence (SEV-2015-0505).
Enzymatic activity and protein corona for atherosclerosis targeting
Enzymatic activity and protein corona drives selective accumulation of nanomicelles in atherosclerosis. Inmunohistochemistry confirms the colocalisation between the enzyme, the ApoB-100 and the nanomicelles, withput significant uptake by macrophages.
Keywords: atherosclerosis, MRI, protein corona, nanomicelles
Antibiotic treatment with Minocycline affects vessel wall remodeling in a murine model of vascular injury (#107)
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 remodeling in response to injury is associated with vascular smooth muscle cell (VSMC) activation and monocyte recruitment that affect extracellular matrix turnover1. Late-gadolinium enhancement (LGE) using Gd-ESMA, an elastin-specific MR contrast agent, has been previously used to quantify cardiovascular diseases5-7 and minocycline, a tetracycline antibiotic, was shown to have cardioprotective properties2-4. We studied (1) the impact of vascular injury and minocycline on vascular remodeling in hyperlipidemic mice using Gd-ESMA and (2) the effect of minocycline on VSMCs and monocytes.
Study design is summarized in Fig.1A. Animal surgical model: Aortic injury was performed as described8. In vivo MRI: All experiments were performed with a 3T Philips Achieva MR scanner equipped with a 47mm single-loop microscopy surface coil. Images were acquired 2h after intravenous administration of Gd-ESMA (0.2mmol/kg). Acquisition parameters are summarised in Fig.1B. Histology: Remodeling was analyzed using Verhoeff Van Gieson elastic staining as well as tropoelastin and ki67 immunohistochemistry (IHC). FACS: Monocyte count was performed using F4/80, CD155 and Ly6C antibodies. In vitro: Migration of primary VSMCs was evaluated by using the scrape-injury model and proliferation using Ki67 immunofluorescence (IF).
Aortic LGE-MR images (Fig 1C) showed increased vascular enhancement (Fig 1D) and R1 relaxation (Fig 1E) in injured mice fed a high-fat-diet (HFD) compared to all other groups. HFD-injured mice treated with minocycline showed decreased enhancement, suggesting a reduction of elastin remodeling (Fig 1D-1E) compared with untreated mice. Histology showed tropoelastin fibers only in the HFD-injured but not in minocycline-treated mice (Fig 1C).
HFD-injured mice demonstrated increased monocyte recruitment compared with HFD only mice (Fig 2A-2B). Fewer monocytes (Fig 2B) and a shift from Ly6Chigh to Ly6Clow subtypes was observed in the minocycline-treated mice (Fig 2C). In vitro studies showed increased VSMC migration when cells were treated with low density lipoproteins (LDL), conditioned M1 macrophage medium or the combination of both and reduced migration when treated with minocycline (Fig 2D-2E). Finally, minocycline reduced VSMC proliferation both in vitro (Fig 2F) and in vivo (Fig 2G).
In vivo MRI showed that elastin remodeling in the abdominal aorta, caused by vascular injury and hyperlipidemia, can be attenuated by minocycline treatment due to its effect on the phenotype of monocytes and VSMCs. Minocycline promotes a shift from Ly6Chigh inflammatory to Ly6Clow reparative monocytes and reduces migration and proliferation of VSMCs. Short-term minocycline treatment thus could be potentially used to reduce vessel wall thickening following vascular intervention because of its anti-proliferative and immunomodulatory properties.
1Dimitry, A. C. Cardiology in review. 2013; 2Ohshima, S. JACC. 2010. 3Shahzad, K. Atherosclerosis. 2011. 4Phinikaridou A. J Am Heart Assoc. 2013. 5Makowski, M.R. Nature Medicine. 2011. 6Botnar, R.M. Circ Cardiovasc Imaging. 2014. 7Protti, A. J Am Heart Assoc. 2015.8Lavin, B. Circ Cardiovasc Imaging. 2015.
The British Heart Foundation (RG/12/1/29262).
(A) Study design. (B) MRI acquisition parameters. (C) 1st row: Representative images of the aortic vessel wall using fused LGE-MRI/MRA; 2nd row: Verhoeff Van Gieson elastic stain of aortas from the different groups; 3rd row: Tropoelastin immunohistochemistry showing fiber deposition (arrows) in the HFD-injury group. (D) LGE-MRI area and (E) relaxation rate (R1) quantification measured by MRI.
(A) Flow cytometry monocyte gating strategy. (B) Quantification of the monocytes and (C) percentage of Ly6Chigh and Ly6Clow in aorta. (D) Quantification of the wound coverage of VSMCs treated with LDL ± minocycline or (E) in combination with M1 macrophages conditioned medium. (F) Quantification of the percentage of VSMC proliferation in vitro by Ki67 IF and (G) in vivo in aortic sections (arrows).
Keywords: MRI, elastin, remodeling, minocycline
Comparison of 18F-FDG and 18F-NaF for non-invasive assessment of plaque progression with PET/CT in a rabbit model of atherosclerosis (#548)
J. Mateo1, C. Velasco2, 3, A. Mota-Cobián2, 3, R. A. Mota4, 5, S. España2, 3
1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Unidad de Imagen Avanzada, Madrid, Spain
Atherosclerosis is a chronic arterial inflammatory disease. Positron emission tomography (PET) provides a non-invasive method to evaluate biological processes that are relevant for disease progression such as arterial inflammation and calcification. 18F-FDG is by far the main radiopharmaceutical tracer used in PET/CT imaging for atherosclerosis detection. The aim of this study was to compare 18F-FDG (marker of inflammation) and 18F-NaF (marker of active calcifications) in order to evaluate the disease progression in a rabbit model of atherosclerosis.
Aortic atherosclerotic lesions were induced in New Zealand white rabbits (n=9) by a combination of atherogenic diet (0.2% cholesterol) and balloon endothelial denudation. Atherosclerotic animals underwent 18F-FDG-PET/CT and 18F-NaF-PET/CT imaging at 5 and 8 months after diet initiation. Two rabbits that were fed standard chow served as healthy controls and provided the basal radiotracer uptake of non-atherosclerotic aorta (baseline). Quantification of radiotracer uptake of the abdominal aorta was determined by measurement of mean standardized uptake values (SUVmean). Ratio paired t-test was used to evaluate the results.
The uptake of both radiotracers was strongly increased in the abdominal aortas of atherosclerotic rabbits in comparison with healthy animals (Fig. 1). However, whereas the uptake of 18F-NaF continued to augment with the progression of the disease (meanSUVmean±SD of 0.24±0.03 at 5 months and 0.43±0.20 at 8 months, p<0.05) the uptake of 18F-FDG showed only a weak increase (meanSUVmean±SD of 0.62±0.13 at 5 months and 0.72±0.14 at 8 months, p=0.12). This indicates that both radiotracers are not interchangeable when quantifying atherosclerotic burden non-invasively.
These results suggest that 18F-FDG and 18F-NaF reflect different biological aspects of the pathogenesis of atherosclerosis, and reveal the importance of using additional tracers such as 18F-NaF for characterizing atherosclerosis development and progression.
Keywords: Atherosclerosis, FDG, NaF, inflammation, active calcification