International Journal of Cardiology 177 (2014) 287–291
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
PET/CT and MR imaging biomarker of lipid-rich plaques using [64Cu]-labeled scavenger receptor (CD68-Fc) Boris Bigalke a,b, Alkystis Phinikaridou a, Marcelo E. Andia a,c, Margaret S. Cooper a, Andreas Schuster a,d,e, Thomas Wurster f, David Onthank g, Götz Münch h, Philip Blower a, Meinrad Gawaz f, Eike Nagel a,i,j,k, Rene M. Botnar a,i,j,k,⁎ a
King's College London, Division of Imaging Sciences and Biomedical Engineering, London, United Kingdom Charité Campus Benjamin Franklin, Universitätsmedizin Berlin, Medizinische Klinik für Kardiologie und Pulmologie, Berlin, Germany c Radiology Department, School of Medicine, Pontificia Universidad Catolica de Chile, Chile d Department of Cardiology and Pulmonology, Georg-August-University, Göttingen, Germany e Department of Cardiology and Pulmonology, German Centre for Cardiovascular Research (DZHK Partner Site), Göttingen, Germany f Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universität Tübingen, Germany g Lantheus Medical Imaging, North Billerica, MA, USA h AdvanceCor GmbH, Martinsried, Germany i BHF Centre of Excellence, King's College London, United Kingdom j Wellcome Trust and EPSRC Medical Engineering Center, King's College London, United Kingdom k NIHR Biomedical Research Centre, King's College London, London, United Kingdom b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Received in revised form 25 August 2014 Accepted 15 September 2014 Available online 22 September 2014 Keywords: 64 Cu-labeled CD68-Fc Atherosclerosis ApoE−/− mice Imaging biomarker of plaque erosion PET/CT and MRI
a b s t r a c t Continued uptake of modified low-density lipoproteins (LDL) by the scavenger receptor, CD68, of activated macrophages is a crucial process in the development of atherosclerotic plaques and leads to the formation of foam cells. Eight-weeks-old male Apolipoprotein E-deficient (ApoE-/-) mice (n=6) were fed a high-fat diet for 12 weeks. C57BL/6J wildtype (WT) mice served as controls (n=6). Positron emission tomography (PET) with an acquisition time of 1800s (NanoPET/CT scanner; Mediso, Hungary & Bioscan, USA) was carried out 24h after intravenous tail vein administration of 50μl 64Cu-CD68-Fc (~20-30μg labeled protein/mouse containing approximately 10-12MBq 64Cu-CD68-Fc per mouse). Three days after PET/CT, all mice received an intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-binding contrast agent to assess plaque burden and vessel wall remodeling. Two hours after injection, mice were imaged in a 3T clinical MR scanner (Philips Healthcare, Best, NL) using a dedicated single loop surface coil (23mm). Enhanced 64Cu-CD68-Fc uptake was found in the aortic arches of ApoE-/- compared to WT mice (ApoE-/- mice:10.5±1.5Bq/cm³ vs. WT mice: 2.1±0.3Bq/cm³; P=0.002). Higher gadolinium-based elastin-binding contrast agent uptake was also detected in the aortic arch of ApoE-/- compared to WT mice using R1 maps (R1=1.47±0.06 s-1 vs. 0.92±0.05 s-1; P b0.001). Radiolabeled scavenger receptor (64Cu-CD68-Fc) may help to target foam cell rich plaques with high content of oxidized LDL. This novel imaging biomarker tool may have potential to identify unstable plaques and for risk stratification. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Oxidative modification of low-density lipoprotein (oxLDL) is a key event in atherosclerosis leading to macrophage-derived formation of lipid-laden foam cells [1,2].
⁎ Corresponding author at: The Rayne Institute, Division of Imaging Sciences & Biomedical Engineering, St Thomas' Hospital, King's College London, Lambeth Palace Rd, London SE1 7EH, United Kingdom. Tel.: +44 2071887242; fax: +44 2071885442. E-mail address: [email protected] (R.M. Botnar).
http://dx.doi.org/10.1016/j.ijcard.2014.09.017 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
Among several different scavenger receptors, the oxLDL binding receptor CD68 is considered as one of the key receptors, which is expressed on the cell surface of macrophages [3]. Previously our group has designed and tested the fusion protein of soluble CD68-Fc to inhibit foam cell formation and to attenuate plaque extension and development of atherosclerosis [4,5]. Thus, the principle of fusion protein administration is not a specific blockade, but a reduced scavenger receptor mediated uptake by the binding of CD68-Fc to its key ligand oxLDL [5]. To develop effective strategies of prevention and therapy, it would be advantageous to visualize lipoproteins and their receptors involved in pathogenesis of atherosclerosis.
288
B. Bigalke et al. / International Journal of Cardiology 177 (2014) 287–291
In pilot studies we showed that radiolabeled CD68-Fc may be a promising imaging biomarker to detect atherosclerotic plaques [6]. However, limited spatial resolution and prolonged half-life time of the originally used 124I may be inappropriate for future translational applications in humans compared to other isotopes of positron emission tomography (PET) with shorter half life time. Based on our experience with 64Cu-radiolabeled glycoprotein VI (GPVI)-Fc as a novel non-invasive molecular imaging biomarker, which may detect subendothelial collagen of plaque erosion and has been tested in the preclinical setting as well as in a recent proof-of-concept study in patients [7,8], we sought to investigate the feasibility of radiolabeling CD68-Fc with 64Cu instead of 124I. The aim of this study was to investigate the merits of the radiolabeled scavenger receptor (64Cu-CD68-Fc) as a novel diagnostic tool for the non-invasive detection of foam cell lipid-rich plaques with high content in oxLDL in the murine model of accelerated atherosclerosis, which may have important preventative and therapeutic implications. 2. Material and methods 2.1. Mouse models Atherosclerosis develops reproducibly in the established ApoE−/− mouse model as previously described [8–10]. Eight-week-old homozygous male apolipoprotein Edeficient (ApoE−/− ) B6.129P2–apoE tm1Unc /J (C57BL/6J background) mice (n = 6) were switched to a high-fat diet (HFD) containing 21% fat from lard, and 0.15% (wt/wt) cholesterol (Special Diets Services, Witham, UK) for 12 weeks. Specific pathogen-free 16-week-old male C57BL/6J wildtype (WT) mice were fed a normal diet and served as controls (n = 6). Both groups of mice (ApoE−/− and WT mice) were purchased from Charles Rivers Laboratories (Edingburgh, UK). Animal studies were performed according to UK Research Councils' and Medical Research Charities' guidelines on Responsibility in the Use of Animals in Bioscience Research, under a UK Home Office License (PPL 70/7097). 2.2. Radiolabeling of the molecular imaging biomarker The basic principle of radiolabeling CD68-Fc as a novel imaging biomarker, which targets oxidized LDL in a similar pattern as the class D (CD68) scavenger receptor of the macrophage foam cell, is schematically shown in Fig. 1A. CD68-Fc protein was conjugated to the bifunctional chelator S-2-(4isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-BnNOTA) as previously described [7,9]. In brief, the protein was stripped of metal ions using ethylenediaminetetraacetic acid and the buffer was exchanged into 0.1 M 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer pH 8.9 by ultracentrifugation. After adding p-SCN-Bn-NOTA (as a 40-fold molar excess to protein) in dimethyl sulfoxide, the conjugation was processed at ambient temperature for 3 h. The conjugated protein was purified and the buffer exchanged into the radiolabeling buffer, 0.1 M ammonium acetate, by ultracentrifugation and stored at − 20 °C for radiolabeling. 64Cu was prepared by 64Ni(p,n)64Cu nuclear reaction on a CTI RDS 112 11 MeV cyclotron. The irradiated 64Ni was dissolved in minimal concentrated hydrochloric acid (100–150 μl) and 64Cu purified to yield 64CuCl2 by loading onto an anion exchange column (Biorad AG1-X8 resin). Excess 64Ni was removed by elution in 9 M HCl. 6 M HCl was then used to raise the pH before elution of the 64CuCl2 in 0.1 M HCl. 64CuCl2 was converted to 64Cu acetate by dilution in 1 M ammonium acetate buffer solution pH 6 prior to radiolabeling. NOTA-CD68-Fc, in 0.1 M ammonium acetate buffer, pH 6, was radiolabeled with 64Cu by addition of 64Cu acetate. The resulting solution was incubated at ambient temperature for 20 min to yield the novel biomarker, 64Cu-CD68-Fc. Assessment of successful radiolabeling was performed by size exclusion HPLC. Radiolabeling experiments for 64 Cu-CD68-Fc achieved a radiochemical purity of 99.5% (Fig. 1B). Cloning of the novel imaging biomarker was performed, partly expressed in Chinese hamster ovary cells as soluble immune adhesin (CD68-Fc) and tested for purity in-vitro (Westernblot, SDS-PAGE). 2.3. PET/CT imaging PET/CT imaging was performed in HFD atherosclerotic ApoE−/− as well as in WT mice according to the methods described previously [7]. All mice received a total activity of ~12 MBq 64Cu-GPVI-Fc by injection into the tail vein followed by delayed (24 h) PET imaging using a NanoPET/CT scanner (Mediso, Budapest, Hungary & Bioscan, Washington DC, USA) with an acquisition time of 1800 s. 2.4. MR imaging To non-invasively assess plaque burden and vascular remodeling in the aortic arch of the ApoE−/− and WT mice magnetic resonance imaging was obtained after administration
of an elastin-specific contrast agent (ESMA), as previously demonstrated in mice and rabbits [7,10–12]. 72 h after PET/CT imaging, all mice were scanned 2 h after intravenous injection of 0.2 mmol/kg bodyweight of ESMA. Delayed enhancement MRI and T1 mapping were performed on a 3T clinical scanner (Philips Healthcare, Best, Netherlands) using a dedicated single loop surface coil (23 mm). Following a 3D-GRE scout scan, contrast enhanced angiography images were acquired for visualization of the aortic arch and the brachiocephalic arteries with a field of view (FOV) = 30 × 30 × 8mm, matrix = 200 × 200, in-plane resolution = 0.15 × 0.15 mm (reconstructed = 0.10 × 0.10 mm), slice thickness = 0.5 mm, TR/TE = 15/6.1 ms and flip angle = 40°. The maximum intensity projection images were used to plan the subsequent delayed enhancement and T1 mapping scans. In order to quantify the ESMA uptake, T1 mapping was performed with the use of a sequence that employs two non-selective inversion pulses with inversion times ranging from 20 to 2000 ms, followed by eight segmented readouts [13]. T1 mapping acquisition parameters included FOV = 36 × 22 × 8mm, matrix = 192 × 102, in-plane resolution = 0.18 × 0.22 mm, measured slice thickness = 0.5 mm, slices = 16, TR/TE = 9.6/4.9 ms, and flip angle = 10° [13]. 2.5. Histology and immunostaining For tissue harvesting, mice were anesthetized with isoflurane, culled with a ketamin/ xylazine overdose and perfused through the left ventricle with 10% formaldehyde for 10 min. Brachiocephalic arteries and aortic arches were processed for paraffin sectioning. Sections were stained with hematoxylin–eosin for cellular infiltration, Miller's elastica van Gieson for elastin and Masson's trichrome for collagen deposition. Immunostaining with a specific antibody was carried out to locate binding of the administered adhesin, CD68-Fc. As previously described [14], serial sections were blocked with H2O2 and 3% BSA (Sigma, Steinheim, Germany), and incubated with a peroxidase-conjugated goat anti-human IgG antibody Fc fragment specific (Jackson Immunoresearch Laboratories Inc., Westgrove, PA, USA) for 1 h at room temperature. Samples were analyzed by a standard microscope (Axiovert 200, Carl Zeiss, Jena, Germany). Quantification of immunostaining for CD68-Fc was processed in ten sections per mouse using digital image analysis system and defined by area fraction (AF) as the ratio of thresholded chromogen and vascular area according to previous description [15]. 2.6. PET/CT and MR imaging analysis PET/CT signals were analyzed using partial volume correction [16]. Quantification of tracer uptake was expressed as standardized uptake value (SUV) by biodistribution from the corrected images according to previous description [7]. To calculate the standardized uptake values, Bq/cm3 was divided by the Becquerels administered (decay corrected to the scan start time) and multiplied by the total body weight of the mouse. Image fusion was performed by proprietary Bioscan InVivoScope software. For quantification of extracellular matrix remodeling by MRI, R1 (R1 = 1/T1) maps were generated using a custom-made software in Matlab (Mathworks, Natick, MA, USA) [17]. 2.7. Statistical analysis Values are presented as mean ± standard deviation. A P-value of b0.05 was considered as statistically significant and data have been evaluated with the Wilcoxon rank sum test. Dunnett's test was performed for posthoc multiple comparisons. Nonparametric Spearman correlation was used to describe associations between two variables. Statistical analysis was performed using SPSS Statistics software for Windows version 19 (IBM SPSS Inc., Chicago, IL, USA).
3. Results The novel imaging biomarker 64Cu-CD68-Fc showed a localized uptake in the aortic arch of ApoE−/− mice on a 12 weeks HFD compared to WT mice on normal diet (Fig. 1C). Using SUV for quantification, this 64Cu-CD68-Fc uptake was found to be significantly enhanced in ApoE−/− compared to WT mice (ApoE−/− mice: 10.5 ± 1.5 Bq/cm3 vs. WT mice: 2.1 ± 0.3 Bq/cm3; P = 0.002) (Fig. 1D). Similar results were found with the gadolinium-based elastin-specific magnetic resonance contrast agent for vessel wall remodeling, which revealed a localized enhancement (Fig. 1E). In agreement with those findings we found higher R1 relaxation rates in the aortic arches of ApoE−/− compared to WT mice (1.47 ± 0.06 s−1 vs. 0.92 ± 0.05 s−1; P b 0.001) (Fig. 1F). R1mapping for assessment of vessel wall remodeling correlated with 64 Cu-CD68-Fc uptake in ApoE−/− mice (rS = 0.946; P = 0.042). Increased oxLDL burden detected by PET/CT imaging and enhanced vascular remodeling on MR imaging in the aortic arch of ApoE−/− compared to WT mice was in agreement with histological and immunohistological findings (Fig. 2A–F). CD68-Fc was detected using
B. Bigalke et al. / International Journal of Cardiology 177 (2014) 287–291
B
A
C
289
D P=0.002
64Cu 15
OxLDL
OxLDL
OxLDL OxLDL
Macrophage Foam Cell
10
ApoE-/- Mouse
15 min
9 6 3
30
CD68 OxLDL
5
35 25
0
20 mAU
CD68
0
SUV [Bq/cm³]
12
cps
CD68-Fc
Wildtype Mice
ApoE-/- Mice
15 10 5 0 0
5
10
Wildtype Mouse
15 min
E
F P
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
PET/CT and MR imaging biomarker of lipid-rich plaques using [64Cu]-labeled scavenger receptor (CD68-Fc) Boris Bigalke a,b, Alkystis Phinikaridou a, Marcelo E. Andia a,c, Margaret S. Cooper a, Andreas Schuster a,d,e, Thomas Wurster f, David Onthank g, Götz Münch h, Philip Blower a, Meinrad Gawaz f, Eike Nagel a,i,j,k, Rene M. Botnar a,i,j,k,⁎ a
King's College London, Division of Imaging Sciences and Biomedical Engineering, London, United Kingdom Charité Campus Benjamin Franklin, Universitätsmedizin Berlin, Medizinische Klinik für Kardiologie und Pulmologie, Berlin, Germany c Radiology Department, School of Medicine, Pontificia Universidad Catolica de Chile, Chile d Department of Cardiology and Pulmonology, Georg-August-University, Göttingen, Germany e Department of Cardiology and Pulmonology, German Centre for Cardiovascular Research (DZHK Partner Site), Göttingen, Germany f Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universität Tübingen, Germany g Lantheus Medical Imaging, North Billerica, MA, USA h AdvanceCor GmbH, Martinsried, Germany i BHF Centre of Excellence, King's College London, United Kingdom j Wellcome Trust and EPSRC Medical Engineering Center, King's College London, United Kingdom k NIHR Biomedical Research Centre, King's College London, London, United Kingdom b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Received in revised form 25 August 2014 Accepted 15 September 2014 Available online 22 September 2014 Keywords: 64 Cu-labeled CD68-Fc Atherosclerosis ApoE−/− mice Imaging biomarker of plaque erosion PET/CT and MRI
a b s t r a c t Continued uptake of modified low-density lipoproteins (LDL) by the scavenger receptor, CD68, of activated macrophages is a crucial process in the development of atherosclerotic plaques and leads to the formation of foam cells. Eight-weeks-old male Apolipoprotein E-deficient (ApoE-/-) mice (n=6) were fed a high-fat diet for 12 weeks. C57BL/6J wildtype (WT) mice served as controls (n=6). Positron emission tomography (PET) with an acquisition time of 1800s (NanoPET/CT scanner; Mediso, Hungary & Bioscan, USA) was carried out 24h after intravenous tail vein administration of 50μl 64Cu-CD68-Fc (~20-30μg labeled protein/mouse containing approximately 10-12MBq 64Cu-CD68-Fc per mouse). Three days after PET/CT, all mice received an intravenous administration of 0.2 mmol/kg body weight of a gadolinium-based elastin-binding contrast agent to assess plaque burden and vessel wall remodeling. Two hours after injection, mice were imaged in a 3T clinical MR scanner (Philips Healthcare, Best, NL) using a dedicated single loop surface coil (23mm). Enhanced 64Cu-CD68-Fc uptake was found in the aortic arches of ApoE-/- compared to WT mice (ApoE-/- mice:10.5±1.5Bq/cm³ vs. WT mice: 2.1±0.3Bq/cm³; P=0.002). Higher gadolinium-based elastin-binding contrast agent uptake was also detected in the aortic arch of ApoE-/- compared to WT mice using R1 maps (R1=1.47±0.06 s-1 vs. 0.92±0.05 s-1; P b0.001). Radiolabeled scavenger receptor (64Cu-CD68-Fc) may help to target foam cell rich plaques with high content of oxidized LDL. This novel imaging biomarker tool may have potential to identify unstable plaques and for risk stratification. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Oxidative modification of low-density lipoprotein (oxLDL) is a key event in atherosclerosis leading to macrophage-derived formation of lipid-laden foam cells [1,2].
⁎ Corresponding author at: The Rayne Institute, Division of Imaging Sciences & Biomedical Engineering, St Thomas' Hospital, King's College London, Lambeth Palace Rd, London SE1 7EH, United Kingdom. Tel.: +44 2071887242; fax: +44 2071885442. E-mail address: [email protected] (R.M. Botnar).
http://dx.doi.org/10.1016/j.ijcard.2014.09.017 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
Among several different scavenger receptors, the oxLDL binding receptor CD68 is considered as one of the key receptors, which is expressed on the cell surface of macrophages [3]. Previously our group has designed and tested the fusion protein of soluble CD68-Fc to inhibit foam cell formation and to attenuate plaque extension and development of atherosclerosis [4,5]. Thus, the principle of fusion protein administration is not a specific blockade, but a reduced scavenger receptor mediated uptake by the binding of CD68-Fc to its key ligand oxLDL [5]. To develop effective strategies of prevention and therapy, it would be advantageous to visualize lipoproteins and their receptors involved in pathogenesis of atherosclerosis.
288
B. Bigalke et al. / International Journal of Cardiology 177 (2014) 287–291
In pilot studies we showed that radiolabeled CD68-Fc may be a promising imaging biomarker to detect atherosclerotic plaques [6]. However, limited spatial resolution and prolonged half-life time of the originally used 124I may be inappropriate for future translational applications in humans compared to other isotopes of positron emission tomography (PET) with shorter half life time. Based on our experience with 64Cu-radiolabeled glycoprotein VI (GPVI)-Fc as a novel non-invasive molecular imaging biomarker, which may detect subendothelial collagen of plaque erosion and has been tested in the preclinical setting as well as in a recent proof-of-concept study in patients [7,8], we sought to investigate the feasibility of radiolabeling CD68-Fc with 64Cu instead of 124I. The aim of this study was to investigate the merits of the radiolabeled scavenger receptor (64Cu-CD68-Fc) as a novel diagnostic tool for the non-invasive detection of foam cell lipid-rich plaques with high content in oxLDL in the murine model of accelerated atherosclerosis, which may have important preventative and therapeutic implications. 2. Material and methods 2.1. Mouse models Atherosclerosis develops reproducibly in the established ApoE−/− mouse model as previously described [8–10]. Eight-week-old homozygous male apolipoprotein Edeficient (ApoE−/− ) B6.129P2–apoE tm1Unc /J (C57BL/6J background) mice (n = 6) were switched to a high-fat diet (HFD) containing 21% fat from lard, and 0.15% (wt/wt) cholesterol (Special Diets Services, Witham, UK) for 12 weeks. Specific pathogen-free 16-week-old male C57BL/6J wildtype (WT) mice were fed a normal diet and served as controls (n = 6). Both groups of mice (ApoE−/− and WT mice) were purchased from Charles Rivers Laboratories (Edingburgh, UK). Animal studies were performed according to UK Research Councils' and Medical Research Charities' guidelines on Responsibility in the Use of Animals in Bioscience Research, under a UK Home Office License (PPL 70/7097). 2.2. Radiolabeling of the molecular imaging biomarker The basic principle of radiolabeling CD68-Fc as a novel imaging biomarker, which targets oxidized LDL in a similar pattern as the class D (CD68) scavenger receptor of the macrophage foam cell, is schematically shown in Fig. 1A. CD68-Fc protein was conjugated to the bifunctional chelator S-2-(4isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-BnNOTA) as previously described [7,9]. In brief, the protein was stripped of metal ions using ethylenediaminetetraacetic acid and the buffer was exchanged into 0.1 M 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer pH 8.9 by ultracentrifugation. After adding p-SCN-Bn-NOTA (as a 40-fold molar excess to protein) in dimethyl sulfoxide, the conjugation was processed at ambient temperature for 3 h. The conjugated protein was purified and the buffer exchanged into the radiolabeling buffer, 0.1 M ammonium acetate, by ultracentrifugation and stored at − 20 °C for radiolabeling. 64Cu was prepared by 64Ni(p,n)64Cu nuclear reaction on a CTI RDS 112 11 MeV cyclotron. The irradiated 64Ni was dissolved in minimal concentrated hydrochloric acid (100–150 μl) and 64Cu purified to yield 64CuCl2 by loading onto an anion exchange column (Biorad AG1-X8 resin). Excess 64Ni was removed by elution in 9 M HCl. 6 M HCl was then used to raise the pH before elution of the 64CuCl2 in 0.1 M HCl. 64CuCl2 was converted to 64Cu acetate by dilution in 1 M ammonium acetate buffer solution pH 6 prior to radiolabeling. NOTA-CD68-Fc, in 0.1 M ammonium acetate buffer, pH 6, was radiolabeled with 64Cu by addition of 64Cu acetate. The resulting solution was incubated at ambient temperature for 20 min to yield the novel biomarker, 64Cu-CD68-Fc. Assessment of successful radiolabeling was performed by size exclusion HPLC. Radiolabeling experiments for 64 Cu-CD68-Fc achieved a radiochemical purity of 99.5% (Fig. 1B). Cloning of the novel imaging biomarker was performed, partly expressed in Chinese hamster ovary cells as soluble immune adhesin (CD68-Fc) and tested for purity in-vitro (Westernblot, SDS-PAGE). 2.3. PET/CT imaging PET/CT imaging was performed in HFD atherosclerotic ApoE−/− as well as in WT mice according to the methods described previously [7]. All mice received a total activity of ~12 MBq 64Cu-GPVI-Fc by injection into the tail vein followed by delayed (24 h) PET imaging using a NanoPET/CT scanner (Mediso, Budapest, Hungary & Bioscan, Washington DC, USA) with an acquisition time of 1800 s. 2.4. MR imaging To non-invasively assess plaque burden and vascular remodeling in the aortic arch of the ApoE−/− and WT mice magnetic resonance imaging was obtained after administration
of an elastin-specific contrast agent (ESMA), as previously demonstrated in mice and rabbits [7,10–12]. 72 h after PET/CT imaging, all mice were scanned 2 h after intravenous injection of 0.2 mmol/kg bodyweight of ESMA. Delayed enhancement MRI and T1 mapping were performed on a 3T clinical scanner (Philips Healthcare, Best, Netherlands) using a dedicated single loop surface coil (23 mm). Following a 3D-GRE scout scan, contrast enhanced angiography images were acquired for visualization of the aortic arch and the brachiocephalic arteries with a field of view (FOV) = 30 × 30 × 8mm, matrix = 200 × 200, in-plane resolution = 0.15 × 0.15 mm (reconstructed = 0.10 × 0.10 mm), slice thickness = 0.5 mm, TR/TE = 15/6.1 ms and flip angle = 40°. The maximum intensity projection images were used to plan the subsequent delayed enhancement and T1 mapping scans. In order to quantify the ESMA uptake, T1 mapping was performed with the use of a sequence that employs two non-selective inversion pulses with inversion times ranging from 20 to 2000 ms, followed by eight segmented readouts [13]. T1 mapping acquisition parameters included FOV = 36 × 22 × 8mm, matrix = 192 × 102, in-plane resolution = 0.18 × 0.22 mm, measured slice thickness = 0.5 mm, slices = 16, TR/TE = 9.6/4.9 ms, and flip angle = 10° [13]. 2.5. Histology and immunostaining For tissue harvesting, mice were anesthetized with isoflurane, culled with a ketamin/ xylazine overdose and perfused through the left ventricle with 10% formaldehyde for 10 min. Brachiocephalic arteries and aortic arches were processed for paraffin sectioning. Sections were stained with hematoxylin–eosin for cellular infiltration, Miller's elastica van Gieson for elastin and Masson's trichrome for collagen deposition. Immunostaining with a specific antibody was carried out to locate binding of the administered adhesin, CD68-Fc. As previously described [14], serial sections were blocked with H2O2 and 3% BSA (Sigma, Steinheim, Germany), and incubated with a peroxidase-conjugated goat anti-human IgG antibody Fc fragment specific (Jackson Immunoresearch Laboratories Inc., Westgrove, PA, USA) for 1 h at room temperature. Samples were analyzed by a standard microscope (Axiovert 200, Carl Zeiss, Jena, Germany). Quantification of immunostaining for CD68-Fc was processed in ten sections per mouse using digital image analysis system and defined by area fraction (AF) as the ratio of thresholded chromogen and vascular area according to previous description [15]. 2.6. PET/CT and MR imaging analysis PET/CT signals were analyzed using partial volume correction [16]. Quantification of tracer uptake was expressed as standardized uptake value (SUV) by biodistribution from the corrected images according to previous description [7]. To calculate the standardized uptake values, Bq/cm3 was divided by the Becquerels administered (decay corrected to the scan start time) and multiplied by the total body weight of the mouse. Image fusion was performed by proprietary Bioscan InVivoScope software. For quantification of extracellular matrix remodeling by MRI, R1 (R1 = 1/T1) maps were generated using a custom-made software in Matlab (Mathworks, Natick, MA, USA) [17]. 2.7. Statistical analysis Values are presented as mean ± standard deviation. A P-value of b0.05 was considered as statistically significant and data have been evaluated with the Wilcoxon rank sum test. Dunnett's test was performed for posthoc multiple comparisons. Nonparametric Spearman correlation was used to describe associations between two variables. Statistical analysis was performed using SPSS Statistics software for Windows version 19 (IBM SPSS Inc., Chicago, IL, USA).
3. Results The novel imaging biomarker 64Cu-CD68-Fc showed a localized uptake in the aortic arch of ApoE−/− mice on a 12 weeks HFD compared to WT mice on normal diet (Fig. 1C). Using SUV for quantification, this 64Cu-CD68-Fc uptake was found to be significantly enhanced in ApoE−/− compared to WT mice (ApoE−/− mice: 10.5 ± 1.5 Bq/cm3 vs. WT mice: 2.1 ± 0.3 Bq/cm3; P = 0.002) (Fig. 1D). Similar results were found with the gadolinium-based elastin-specific magnetic resonance contrast agent for vessel wall remodeling, which revealed a localized enhancement (Fig. 1E). In agreement with those findings we found higher R1 relaxation rates in the aortic arches of ApoE−/− compared to WT mice (1.47 ± 0.06 s−1 vs. 0.92 ± 0.05 s−1; P b 0.001) (Fig. 1F). R1mapping for assessment of vessel wall remodeling correlated with 64 Cu-CD68-Fc uptake in ApoE−/− mice (rS = 0.946; P = 0.042). Increased oxLDL burden detected by PET/CT imaging and enhanced vascular remodeling on MR imaging in the aortic arch of ApoE−/− compared to WT mice was in agreement with histological and immunohistological findings (Fig. 2A–F). CD68-Fc was detected using
B. Bigalke et al. / International Journal of Cardiology 177 (2014) 287–291
B
A
C
289
D P=0.002
64Cu 15
OxLDL
OxLDL
OxLDL OxLDL
Macrophage Foam Cell
10
ApoE-/- Mouse
15 min
9 6 3
30
CD68 OxLDL
5
35 25
0
20 mAU
CD68
0
SUV [Bq/cm³]
12
cps
CD68-Fc
Wildtype Mice
ApoE-/- Mice
15 10 5 0 0
5
10
Wildtype Mouse
15 min
E
F P
