Open Access

αVβ3 integrin-targeted microSPECT/CT imaging of inflamed atherosclerotic plaques in mice

  • David Vancraeynest1, 5Email author,
  • Véronique Roelants2,
  • Caroline Bouzin3,
  • François-Xavier Hanin2,
  • Stephan Walrand2,
  • Vanesa Bol2,
  • Anne Bol2,
  • Anne-Catherine Pouleur1, 5,
  • Agnès Pasquet1, 5,
  • Bernhard Gerber1, 5,
  • Philippe Lesnik4,
  • Thierry Huby4,
  • François Jamar2 and
  • Jean-Louis Vanoverschelde1, 5
EJNMMI Research20166:29

https://doi.org/10.1186/s13550-016-0184-9

Received: 19 January 2016

Accepted: 15 March 2016

Published: 24 March 2016

Abstract

Background

αVβ3-integrin is expressed by activated endothelial cells and macrophages in atherosclerotic plaques and may represent a valuable marker of high-risk plaques. We evaluated 99mTc-maraciclatide, an integrin-specific tracer, for imaging vascular inflammation in atherosclerotic lesions in mice.

Methods

Apolipoprotein E-negative (ApoE−/−) mice on a Western diet (n = 10) and normally fed adult C57BL/6 control mice (n = 4) were injected with 99mTc-maraciclatide (51.8 ± 3.7 MBq). A blocking peptide was infused in three ApoE−/− mice; this condition served as another control. After 90 min, the animals were imaged via single-photon emission computed tomography (SPECT). While maintained in the same position, the mice were transferred to computed tomography (CT) to obtain contrast-enhanced images of the aortic arch. Images from both modalities were fused, and signal was quantified in the aortic arch and in the vena cava for subtraction of blood-pool activity. The aorta was carefully dissected after imaging for gamma counting, autoradiography, and histology.

Results

Tracer uptake was significantly higher in ApoE−/− mice than in both groups of control mice (1.56 ± 0.33 vs. 0.82 ± 0.24 vs. 0.98 ± 0.11, respectively; P = 0.006). Furthermore, higher tracer activity was detected via gamma counting in the aorta of hypercholesterolemic mice than in both groups of control mice (1.52 ± 0.43 vs. 0.78 ± 0.19 vs. 0.47 ± 0.31 99mTc-maraciclatide %ID/g, respectively; P = 0.018). Autoradiography showed significantly higher tracer uptake in the atherosclerotic aorta than in the control aorta (P = 0.026). Finally, in the atherosclerotic aorta, immunostaining indicated that the integrin signal came predominantly from macrophages and was correlated with the macrophage CD68 immunomarker (r = 0.73).

Conclusions

99mTc-maraciclatide allows in vivo detection of inflamed atherosclerotic plaques in mice and may represent a non-invasive approach for identifying high-risk plaques in patients.

Keywords

αVβ3 integrinMaraciclatideVulnerable atherosclerotic plaqueSPECT imaging

Background

In the majority of cases, acute vascular events such as acute coronary syndrome or stroke are caused by the disruption of a vulnerable atherosclerotic lesion [1]. Compared to stable plaques, “rupture-prone” or “vulnerable” plaques regularly exhibit several features, such as inflammation and neoangiogenesis [2]. Identifying atherosclerotic plaques before they rupture should therefore represent a major advance in the management of atherosclerotic disease. Accordingly, invasive and non-invasive imaging techniques are under development for accurately detecting vulnerable plaques [3, 4].

The αVβ3 integrin is a ubiquitous receptor that is expressed on a variety of cell types; it interacts with ligands present in the extracellular matrix or expressed on the cell surface. This integrin plays a role in diverse biological processes. αVβ3 integrin is detected in situ on macrophages in early and advanced atherosclerotic lesions and could regulate macrophage functional maturation into foam cells [5]. In atherosclerotic plaques, both inflammatory cells (monocytes and macrophages) and activated endothelial cells associated with neoangiogenesis can express αVβ3 integrin [6]. Therefore, αVβ3 expression, which is a combined marker of both inflammation and angiogenesis, may represent a useful imaging target for assessing plaque vulnerability. Several tracers for positron emission tomography (PET) and magnetic resonance imaging (MRI) that display highly specific binding to the αVβ3 integrin have been successfully tested in animal models of vascular inflammation [79] and in human carotid atherosclerosis [10].

99mTc-maraciclatide (GE Healthcare, Amersham, UK) is a cyclic peptide that contains an arginine-glycine-aspartic acid (RGD) tripeptide sequence with a high affinity for vitronectin αVβ3 integrin receptors [11]. This single-photon emission computed tomography (SPECT) tracer has been shown to localize to inflammatory infiltrate associated with angiogenesis in a murine model of hindlimb ischemia-induced angiogenesis [12, 13] and in angiogenesis induced by local IGF-1 expression after myocardial infarction [14]. More recently, maraciclatide uptake was detectable by SPECT/computed tomography (CT) in chemically injured carotid arteries in mice [15]. The potential for imaging αVβ3 integrin expression in atherosclerotic plaques with 99mTc-maraciclatide in spontaneous atherosclerotic mice remains unknown.

In the present study, we sought to evaluate 99mTc-maraciclatide for imaging vascular inflammation by studying its in vivo uptake in the atherosclerotic mouse aorta. Uptake of the tracer in the vessel wall was validated via gamma counting and autoradiography. Specificity was confirmed with competition experiments.

Methods

Animal model and experimental design

The study group consisted of 4- to 8-week-old male apolipoprotein E (ApoE)−/− mice (n = 10) acquired from Taconic Europe (Lille Skensved, Denmark) and 4- to 8-week-old male C57BL/6 control mice (n = 4). ApoE−/− mice were kept on a Western diet (1.25 % cholesterol, 16 % cocoa butter, U8220 version 97, Safe, Augy, France) for 26 weeks preceding the examinations to induce atherosclerosis. C57BL/6 mice were fed normally for the same 26 weeks. Cholesterol-fed ApoE−/− mice were divided into two groups, each of which received either (1) 99mTc-maraciclatide (n = 7) or (2) pretreatment with blocking peptide (50 μg of NC100717, unlabeled precursor, GE Healthcare, Amersham, UK) 2 min before delivery of 99mTc-maraciclatide (n = 3). The latter group was designed to demonstrate imaging specificity. All control-diet C57BL/6 mice received 99mTc-maraciclatide (n = 4) (Fig. 1). The experimental protocol was approved by the Institutional Animal Studies Committee of the Université Catholique de Louvain.
Fig. 1

Experimental design

MicroSPECT and CT imaging

Mice were anesthetized with 1–3 % isoflurane, and the right femoral vein was isolated for placement of a catheter (27-g butterfly needle with 12-cm polyurethane tubing, Visualsonics, Toronto, Canada) to facilitate the injection of radiotracers and contrast medium. All animals were injected intravenously with 51.8 ± 3.7 MBq of 99mTc-maraciclatide, a 99mTc-labeled chelated peptide conjugate containing an RGD motif targeting αV integrin. Anesthesia was maintained, and each mouse was put into a sarcophagus especially designed to keep the animal in the same position throughout the experiments. A tube containing free 99mTc was fixed around the sarcophagus to allow perfect fusion between SPECT images and CT images. SPECT was performed 90 min after radiotracer injection using a high-resolution small-animal imaging microSPECT system [16] (Linoview SPECT system, The Netherlands) equipped with 0.4-mm-wide slit-slat collimators. The four detectors followed four linear orbits surrounding the animal, providing linograms for a total acquisition time of 30 min with a 35 % energy window centered at 140 keV. The resulting linograms were reconstructed using the expectation maximization maximum likelihood algorithm without attenuation or resolution correction. The system spatial resolution was 0.6 mm resulting from the collimator slit width and the crystal pixel size. The sarcophagus was then transferred to a 16-detector CT system (Philips Medical System, The Netherlands). Animals were injected with a bolus infusion of CT contrast (150 μL iodinated CT contrast diluted 1:2, Iomeron 400, Bracco, UK) over 5 s, and CT imaging was performed 2 s after the start of bolus injection to precisely identify arterial structures. The following parameters were used: tube rotation speed, 420 ms; detector collimation, 16 × 0.75 mm; tube voltage, 140 kV; and effective tube current, 400 mAs.

Images from both modalities were subsequently rigidly registered using PMOD version 2.65 (PMOD Technologies, Ltd., Adliswil, Switzerland). The volume data were analyzed using the conventional transverse, coronal, and sagittal views. For quantitative analysis of tracer uptake into the plaque, a 3D region of interest (ROI) was drawn manually at the level of the aortic arch on the CT transverse images and copied to the co-registered SPECT images The totality of the arch was included in the analysis. The size of the mice vena cava being sufficient to avoid partial volume effect, a ROI in the inferior vena cava was used to determine background activity accurately. Averaged counts in each ROI were used to calculate the target to background ratio (TBR) defined as the aortic arch activity (counts/voxel) divided by the vena cava activity (counts/voxel).

Gamma well counting

Mice were euthanized immediately after in vivo SPECT/CT. The aortic arch and the descending aorta of each animal were carefully dissected and washed in saline solution. Tissue samples were weighed. Tissue 99mTc activity was measured using a gamma well counter (Packard COBRA II gamma counter) with an appropriate energy window (140 keV) and corrected for background, decay time, and tissue weight. Corrected counts were converted to μCi per milligram of tissue by using a previously determined counter efficiency. Activity was calculated as percentage of injected dose per weight (%ID/g).

Autoradiography

Aortic samples from ApoE−/− mice (n = 3) and from C57BL/6 control mice (n = 3) were exposed to an imaging plate (Fuji Imaging Plate, Fuji Photo Film Co. Ltd., Japan) immediately after gamma counting. After overnight exposure, the imaging plates were scanned with the Fuji Analyzer FLA-2000. The images were analyzed for count densities (quantification level/mm2) with image analysis software (AIDA Image Analyzer, Raytest, Isotopenmessgeraete GmbH, Germany) by drawing regions of interest on the aortic arch.

Immunostaining

Aortic samples from three ApoE−/− mice and from one C57BL/6 control mouse were embedded in optimum cutting temperature compound, snap-frozen, and stored at −80 °C. Histology was performed on 5 μm-thick cryosections of the aortic arch. Macrophages and endothelial cells were identified with rat monoclonal IgG (respectively: rat anti-mouse CD68, product MCA1957GA, diluted 1:50, AbD Serotec; rat anti-mouse CD31, product 550274, diluted 1:50, BD Biosciences). αV integrin was identified with rabbit anti-mouse CD51 (product 210-537-R100, polyclonal, diluted 1:1000, Enzo Life Sciences). Biotinylated Alexa 568 (red) anti-rabbit or Alexa 488 (green) anti-rat IgG antibodies were used as secondary reagents. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Images were acquired and digitized under high power (×63 oil-immersion objective) with a Zeiss AxioImager.z1 microscope. Signals from CD68, CD51, and CD31 were quantified with ImageJ (open-source software developed by the National Institutes of Health) and expressed as the percent positive area out of the total tissue area.

Statistical analysis

All results are expressed as mean ± standard deviation. Continuous variables were compared among the three groups of mice using Kruskal-Wallis analysis of variance. Individual comparisons between groups were evaluated post hoc using the Mann-Whitney test with Bonferroni adjustment for multiple testing. Associations between any two variables were addressed using Pearson’s correlation. P < 0.05 was considered statistically significant. The statistical analyses were performed using SPSS version 1.5.0 statistical software (Chicago, IL).

Results

All ApoE−/− mice had atherosclerotic plaques predominantly located at the level of the aortic arch and the supra-aortic vessels (Fig. 2a). As expected, no aortic plaques were found in C57BL/6 control mice (Fig. 2b).
Fig. 2

Representative images of the macroscopic appearance of the aorta of a cholesterol-fed ApoE−/− mouse (a) and a normally fed C57BL/6 control mouse (b). Note the presence of atherosclerotic plaques predominantly located at the level of the aortic arch and the supra-aortic vessels of the hyperlipidemic mouse (white arrows); no plaque is apparent in the aorta of the control mouse

MicroSPECT/CT in vivo imaging of αVβ3 integrin in the aortic arch

ApoE−/− mice and control mice underwent 99mTc-maraciclatide microSPECT at 30 ± 4 weeks. Increased tracer uptake was readily visible on the aortic arch of ApoE−/− mice that did not receive the blocking peptide (Fig. 3a). In contrast, no signal was detected in the aortic arch of C57BL/6 control mice (Fig. 3b). As expected, no signal was visible in the aortic arch of ApoE−/− mice that received the blocking peptide 2 min before 99mTc-maraciclatide injection (Fig. 3c).
Fig. 3

This figure shows coronal, axial, and sagittal views from CT (left), fused microSPECT/CT (middle), and SPECT (right) of αVβ3 integrin imaging, respectively. MicroSPECT/CT allows imaging of 99mTc-maraciclatide uptake (red arrow) into atherosclerotic plaques of ApoE−/− mouse. a CT images, SPECT/CT overlay images, and SPECT images obtained in an ApoE−/− mouse. b CT images, SPECT/CT overlay images, and SPECT images obtained in a C57BL/6 control mouse. c CT images, SPECT/CT overlay images, and SPECT images obtained in an ApoE−/− mouse who received the blocking peptide. Animals were kept under gas anesthesia (1–3 % isoflurane in O2 at a flow rate of 2 L/min). SPECT was performed 90 min after injection of about 52 MBq of 99mTc-maraciclatide. Total acquisition time was 30 min. The focal thyroid free-99mTc uptake is marked by an asterisk. LV left ventricle, AA aortic arch, CPV counts per voxel

Tracer uptake was quantified in the aortic arch of all animals. Although the signal-to-noise ratio of 99mTc-maraciclatide was not very high, quantitative analysis from in vivo images indicated significantly higher uptake, expressed in TBR, in the aorta of ApoE−/− mice, as compared with C57BL/6 control mice and with ApoE−/− mice that received the blocking peptide 2 min before 99mTc-maraciclatide injection (1.56 ± 0.33 vs. 0.82 ± 0.24 vs. 0.98 ± 0.11, respectively; P = 0.006; Fig. 4). These data establish the specificity of tracer uptake.
Fig. 4

MicroSPECT-derived quantification of 99mTc-maraciclatide uptake in the aortic arch of ApoE−/− mice (n = 7), C57BL/6 control mice (n = 4), and ApoE−/− mice who received the blocking peptide immediately before 99mTc-maraciclatide injection (n = 3). P value on top of the graph indicates the Kruskal-Wallis test result for the three experimental groups. Asterisks indicate significant difference, according to the Mann-Whitney test for independent samples with Bonferroni adjustment for multiple testing; *P < 0.05, **P < 0.01. Number sign indicates a P value >0.05. Ctrl control

Validation of 99mTc-maraciclatide in vivo uptake with gamma counting

The significant increase in 99mTc-maraciclatide uptake within the aortic arch of ApoE−/− mice that was observed with microSPECT imaging in vivo was confirmed with gamma counting. 99mTc-maraciclatide microSPECT uptake significantly correlated with tracer activity measured by gamma counting (r = 0.63, P = 0.038). Higher tracer activity was detected in the aortas from ApoE−/− mice than in the aortas from C57BL/6 control mice and in those from ApoE−/− mice that received the blocking peptide (1.52 ± 0.43 vs. 0.78 ± 0.19 vs. 0.47 ± 0.31 99mTc-maraciclatide %ID/g, respectively; P = 0.018; Fig. 5).
Fig. 5

Counts obtained by gamma counting of the aortic samples of ApoE−/− mice (n = 7), C57BL/6 control mice (n = 4), and ApoE−/− mice who received the blocking peptide immediately before 99mTc-maraciclatide injection (n = 3). Data are expressed as percentage of the injected dose of 99mTc-maraciclatide per gram (%ID/g). P value on top of the graph indicates the Kruskal-Wallis test result for the three experimental groups. Asterisks indicate significant difference, according to the Mann-Whitney test for independent samples with Bonferroni adjustment for multiple testing; *P < 0.05. Number sign indicates a P value >0.05. Ctrl control

Validation of 99mTc-maraciclatide in vivo uptake with autoradiography

To confirm the ability of 99mTc-maraciclatide to detect αVβ3 integrin expression in vivo, aortic arches from ApoE−/− mice (n = 3) and from C57BL/6 control mice (n = 3) were harvested for autoradiography. Consistent with our microSPECT and gamma counting analyses, uptake in hyperlipidemic aortas was visibly higher than in control aortas (Fig. 6a, b). A region of interest was drawn around the entire aortic cross of the animals, and the relative autoradiographic intensity was measured. Again, the highest uptake (quantification level/mm2) was identified in the aortas from ApoE−/− mice (P = 0.026; Fig. 6c).
Fig. 6

Autoradiographic analysis of 99mTc-maraciclatide uptake. 99mTc-maraciclatide uptake is obvious in the aorta of the ApoE−/− mouse (a) while a minimal tracer uptake corresponding to background activity is identified in the aorta of the C57BL/6 control mouse (b). Higher tracer uptake in the aortas of ApoE−/− mice was confirmed by drawing regions of interest around the aortic cross (c). P value on top of the graph indicates the Mann-Whitney test result between the two experimental groups. Ctrl control

Immunohistology

Many types of cells in atherosclerotic plaques, including macrophages, endothelial cells, and vascular smooth muscle cells, may express αV integrin. However, in our model, αV integrin was mostly expressed by macrophages. Indeed, we observed a significant correlation between the patterns of CD68 and CD51 expression (r = 0.73, P = 0.04; Fig. 7a). A representative micrograph of the co-labeling of macrophages expressing CD68 and CD51 is shown in Fig. 7b–e.
Fig. 7

Immunostaining of inflammation and αV integrin. a Significant correlation between percent area of CD68 staining and percent area of CD51 staining (Pearson’s r = 0.73, P = 0.04). Histopathological characterization of the aortic tissue section from an atherosclerotic animal. b Merged image of CD68 (macrophages in green) and CD51 (αV integrin in red) co-labeling. High-power views (corresponding to the box in b) showing representative micrographs of co-labeling (c), macrophage (d), and αV integrin (e). Arrows point to cells positive for both markers. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue) (c). Scale bar = 20 μm in b. Scale bar = 10 μm in ce

In contrast, cells expressing both CD31 and CD51 which were supposed to be neo-endothelial cells were very rarely identified in the thickness of the plaque (Fig. 8). We did not detect any correlation between percent staining for CD31 and CD51 (r = 0.31, P = 0.41).
Fig. 8

Histopathological characterization of the aortic tissue section from an atherosclerotic animal. a Merged image of CD31 (microvessels in green) and CD51 (αV integrin in red) co-labeling. High-power views (corresponding to the box in a) showing representative micrographs of co-labeling (b), microvessels (c), and αV integrin (d). Arrows point to cells positive for both markers. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue) (b). Scale bar = 20 μm in a. Scale bar = 10 μm in bd

Discussion

Integrins belong to a group of cell-adhesion molecules; they are heterodimeric transmembrane glycoproteins that play a role in cell-cell and cell-matrix interactions [17]. Because both activated macrophages and endothelial cells can express high levels of integrin, especially αVβ3 integrin, αVβ3 expression represents a combined marker of inflammation and angiogenesis, which are both implicated in plaque vulnerability. αVβ3 integrin binds extracellular-matrix proteins via the RGD sequence, which is present in the 99mTc-maraciclatide compound. 99mTc-maraciclatide has favorable kinetic properties for imaging, such as high affinity for integrin receptors, high metabolic stability in the circulation, and rapid renal excretion [11]. Our results demonstrate the potential for imaging αVβ3 integrin expression with 99mTc-maraciclatide and microSPECT/CT in inflamed plaques of atherosclerotic mice. We validated our results with gamma counting, autoradiography, and histology. Furthermore, competition experiments confirmed the specificity of the signal.

Atherosclerotic plaque angiogenesis has been imaged using αVβ3 integrin-targeted nanoparticles and MRI [9)], and vascular inflammation has been successfully evaluated with αVβ3 integrin-targeted PET [8, 10]. Here, we demonstrated that atherosclerotic lesions can be imaged with a 99mTc tracer and a SPECT system, a strategy that confers several advantages. First, SPECT imaging is much more widely available and less expensive than PET or MRI. Second, the 99mTc radionuclide is easier to prepare and easier to use than many PET tracers that are usually labeled with short-lived radioisotopes. Finally, like other interesting tracers (mainly PET tracers) [18, 19], 99mTc-maraciclatide could be used for coronary imaging, a major area of interest. This is not the case for 18F-fluoro-2-deoxy-D-glucose (FDG), which is currently the most validated PET tracer for arterial inflammation imaging in humans [20]. Indeed, coronary artery imaging with that tracer is more challenging than carotid imaging, mainly due to intense FDG uptake in adjacent myocardium. Even if adequate preparation of patients allows minimal myocardial FDG uptake [21], FDG utilization in imaging of coronary wall inflammation remains to be validated; some data suggest that its utilization in coronary imaging is inappropriate [19]. Tracers like 99mTc-maraciclatide that could overcome this limitation would constitute a remarkable step forward for coronary artery imaging. However, given the relative low uptake of the tracer and the spatial resolution of clinical SPECT (6.7–15.3 mm) [22], the fact that 99mTc-maraciclatide is a SPECT tracer could be perceived as a limitation for its widespread use in human arterial inflammation imaging, especially for coronary artery imaging. Nevertheless, it is unlikely that in the future, vulnerable plaque identification will be based on a single imaging modality. Multimodality imaging will be probably necessary. The combined assessment of anatomical markers of high-risk plaques (soft plaques, spotty calcifications, eccentric remodeling) which can be assessed by contrast-enhanced CT with functional markers like inflammation and neoangiogenesis which can be assessed by 99mTc-maraciclatide SPECT/CT could for instance represent an interesting dual approach for identifying vulnerable plaque. For this study, we used a preclinical animal scanner with a much higher spatial resolution than clinical SPECT systems [16]. Although this scanner enables sub-millimeter resolution, microscopic abdominal atherosclerosis was not visualized in our animal model. Only macroscopic plaques, such as those visually identified in the aortic arch (Fig. 2), were imaged with 99mTc-maraciclatide, suggesting that a “critical mass” is needed for plaque imaging. Whether the combined use of 99mTc-maraciclatide and clinical SPECT scanners will enable the detection of inflamed atherosclerotic plaques in humans remains unknown. However, cooperation between researchers and manufacturers will ensure that SPECT/CT becomes a more accurate and reliable tool in the next decade.

The plaques in our ApoE-deficient mice are “human-like,” as they are very similar to plaques observed in patients [23]. Furthermore, in ApoE−/− mice like those we used in this work, plaques which are present at the origin of the great vessels of the neck and on which we concentrated in this study (Fig. 2) demonstrate features likely to be the “murine parallel” of those in vulnerable human plaques [24]. We therefore believe that this tracer may be of interest for detecting high-risk plaques in humans and perhaps also in tracking the effect of aggressive pharmacological treatment, like high dose of statins. Furthermore, this tracer has been approved for human use; its safety and tolerance have already been successfully tested in patients with breast cancer [25]. Therefore, it would be interesting to move from preclinical animal studies to clinical studies in order to test this tracer in the field of human atherosclerosis.

We detected a strong correlation between immunostaining for αV integrin (CD51) and for macrophages (CD68); in contrast, co-staining for αV integrin and neovessels (CD31) identified very few cells that were positive for both immunomakers. This finding is similar to observations reported by previous studies [8, 15, 26]. It also suggests that the 99mTc-maraciclatide signal largely results from inflammatory infiltrate rather than from neoangiogenesis per se. Inflammation plays a key role in atherosclerosis and is linked to plaque vulnerability. The presence of inflammation in the arterial wall of large arteries measured by FDG-PET/CT has been shown to predict acute clinical events [27]. 99mTc-maraciclatide could thus help to refine the clinician’s ability to stratify patient risk for acute events such as stroke, sudden cardiac death, or acute coronary syndromes.

Some limitations must be acknowledged for this study. First, the number of animals is small. Since it was unknown whether reliable SPECT signal could be obtained from the atherosclerotic lesions of mice, this study was designed as a “feasibility study.” Nonetheless, tracer uptake was clearly detected in the aortic arch of atherosclerotic mice in comparison with control animals. Furthermore, the imaging results were validated with several other techniques, including gamma counting and autoradiography, and blocking with excess unlabeled precursor confirmed the specificity of the SPECT signal. Finally, the number of animals used in this study is similar to that used in other comparable studies [8, 26]. As a second limitation, we did not measure mRNA expression of the genes encoding CD68, CD31, or CD51 in mouse aortas via quantitative reverse transcription polymerase chain reaction. Nevertheless, we obtained convincing images with immunohistology, and the quantification of the various markers revealed a clear relationship between integrin expression and macrophages. The co-expression of integrin and CD31 was clearly weaker. These results are consistent with those obtained by Yoo et al [26].

Conclusions

Here, we have demonstrated that 99mTc-maraciclatide SPECT/CT specifically detects αVβ3 integrin expression in inflamed atherosclerotic plaques in ApoE−/− mice. This tracer may thus represent an interesting non-invasive approach for identifying high-risk plaques in human patients and for monitoring their global risk for presenting acute vascular events. Its clinical value must be defined by future prospective human studies.

Declarations

Funding sources

This work was supported in part by the Fonds de la Recherche Scientifique Médicale – F.N.R.S. (n° 3.4592.09), Brussels, Belgium. DV is supported by the Fonds de Recherche Clinique of the Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Pôle de Recherche Cardiovasculaire (CARD), Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain
(2)
Pôle d’Imagerie Médicale, Radiothérapie et Oncologie (MIRO), Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain
(3)
IREC Imaging Platform, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain
(4)
INSERM UMR_S 1166, Integrative Biology of Atherosclerosis Team, Université Pierre et Marie Curie-Paris6 and institute of Cardiometabolism and Nutrition (ICAN), Pitié-Salpêtrière Hospital
(5)
Division of Cardiology, Cliniques Universitaires St-Luc

References

  1. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation. 1995;92:657–71.View ArticlePubMedGoogle Scholar
  2. Virmani R, Burke AP, Farb A, Kolodgie FD. Pathology of the vulnerable plaque. J Am Coll Cardiol. 2006;47:C13–8.View ArticlePubMedGoogle Scholar
  3. Narula J, Garg P, Achenbach S, Motoyama S, Virmani R, Strauss HW. Arithmetic of vulnerable plaques for noninvasive imaging. Nat Clin Pract Cardiovasc Med. 2008;5 Suppl 2:S2–10.View ArticlePubMedGoogle Scholar
  4. Vancraeynest D, Pasquet A, Roelants V, Gerber BL, Vanoverschelde JL. Imaging the vulnerable plaque. J Am Coll Cardiol. 2011;57:1961–79.View ArticlePubMedGoogle Scholar
  5. Antonov AS, Kolodgie FD, Munn DH, Gerrity RG. Regulation of macrophage foam cell formation by αVβ3 integrin. Potential role in human atherosclerosis. Am J Pathol. 2004;165:247–58.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Hoshiga M, Alpers CE, Smith LL, Giachelli CM, Schwartz SM. αVβ3 integrin expression in normal and atherosclerotic artery. Circ Res. 1995;77:1129–35.View ArticlePubMedGoogle Scholar
  7. Burtea C, Laurent S, Murariu O, Rattat D, Toubeau G, Verbruggen A, et al. Molecular imaging of αVβ3 integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA. Cardiovasc Res. 2008;78:148–57.View ArticlePubMedGoogle Scholar
  8. Laitinen I, Saraste A, Weidl E, Poethko T, Weber AW, Nekolla SG, et al. Evaluation of αVβ3 integrin-targeted positron emission tomography tracer 18F-galacto-RGD for imaging of vascular inflammation in atherosclerotic mice. Circ Cardiovasc Imaging. 2009;2:331–8.View ArticlePubMedGoogle Scholar
  9. Winter PM, Morawski AM, Caruthers SD, Fuhrhop RW, Zhang H, Williams TA, et al. Molecular imaging of angiogenesis in early-stage atherosclerosis with αVβ3-integrin-targeted nanoparticles. Circulation. 2003;108:2270–4.View ArticlePubMedGoogle Scholar
  10. Beer AJ, Pelisek J, Heider P, Saraste A, Reeps C, Metz S, et al. PET/CT imaging of integrin αVβ3 expression in human carotid atherosclerosis. JACC Cardiovasc Imaging. 2014;7:178–87.View ArticlePubMedGoogle Scholar
  11. Edwards D, Jones P, Haramis H, Battle M, Lear R, Barnett DJ, et al. 99mTc-NC100692—a tracer for imaging vitronectin receptors associated with angiogenesis: a preclinical investigation. Nucl Med and Biol. 2008;32:365–75.View ArticleGoogle Scholar
  12. Hua J, Dobrucki LW, Sadeghi MM, Zhang J, Bourke BN, Cavaliere P, et al. Noninvasive imaging of angiogenesis with a 99mTc-labeled peptide targeted at αVβ3 integrin after murine hindlimb ischemia. Circulation. 2005;111:3255–60.View ArticlePubMedGoogle Scholar
  13. Dobrucki LW, Dione DP, Kalinowski L, Dione D, Mendizabal M, Yu J, et al. Serial noninvasive targeted imaging of peripheral angiogenesis: validation and application of a semiautomated quantitative approach. J Nucl Med. 2009;50:1356–63.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Dobrucki LW, Tsutsumi Y, Kalinowski L, Dean J, Gavin M, Sen S, et al. Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging. J Mol Cell Cardiol. 2010;48:1071–9.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Razavian M, Marfatia R, Mongue-Din H, Tavakoli S, Sinusas AJ, Zjang J, et al. Integrin-targeted imaging of inflammation in vascular remodelling. Arterioscler Thromb Vasc Biol. 2011;31:2820–6.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Walrand S, Jamar F, de Jong M, Pauwels S. Evaluation of novel whole-body high-resolution rodent SPECT (Linoview) based on direct acquisition of linogram projections. J Nucl Med. 2005;46:1872–80.PubMedGoogle Scholar
  17. Beer AJ, Schwaiger M. Imaging of integrin αVβ3 expression. Cancer Metastasis Rev. 2008;27:631–44.View ArticlePubMedGoogle Scholar
  18. Gaemperli O, Shalhoub J, Owen DR, Lamare F, Johansson S, Fouladi N, et al. Imaging intraplaque inflammation in carotid atherosclerosis with 11C-PK11195 positron emission tomography/computed tomography. Eur Heart J. 2012;33:1902–10.View ArticlePubMedGoogle Scholar
  19. Joshi NV, Vesey AT, Williams MC, Shah AS, Calvert PA, Craighead FH, et al. 18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet. 2014;383:705–13.View ArticlePubMedGoogle Scholar
  20. Figueroa AL, Abdelbaky A, Truong QA, Corsini E, MacNabb MH, Lavender ZR, et al. Measurement of arterial activity on routine FDG PET/CT images improves prediction of risk of future CV events. JACC Cardiovasc Imaging. 2013;6:1250–59.View ArticlePubMedGoogle Scholar
  21. Demeure F, Hanin FX, Bol A, Vincent MF, Pouleur AC, Gerber B, et al. A randomized trial on the optimization of 18F-FDG myocardial uptake suppression: implications for vulnerable coronary plaque imaging. J Nucl Med. 2014;55:1629–35.View ArticlePubMedGoogle Scholar
  22. Slomka PJ, Berman DS, Germano G. New cardiac cameras: single-photon emission CT and PET. Semin Nucl Med. 2014;44(4):232–51.View ArticlePubMedGoogle Scholar
  23. Pendse AA, Arbones-Mainar JM, Johnson LA, Altenburg MK, Maeda N. Apolipoprotein E knock-out and knock-in mice: atherosclerosis, metabolic syndrome, and beyond. J Lipid Res. 2009;50:S178–82.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Meir KS, Leitersdorf E. Atherosclerosis in the apolipoprotein E-deficient mouse. A decade of progress. Arterioscler Thromb Vasc Biol. 2004;24:1006–14.View ArticlePubMedGoogle Scholar
  25. Bach-Gansmo T, Danielsson R, Saracco A, Wilczek B, Bogsrud TV, Fangberget A, et al. Integrin receptor imaging of breast cancer: a proof-of-concept study to evaluate 99m Tc-NC100692. J Nucl Med. 2006;47:1434–9.PubMedGoogle Scholar
  26. Yoo JS, Lee J, Jung JH, Moon BS, Kim S, Lee BC, Kim SE. SPECT/CT imaging of high-risk atherosclerotic plaques using integrin-binding RGD dimer peptides. Sci Rep. 2015;5:11752.View ArticlePubMedGoogle Scholar
  27. Rominger A, Saam T, Wolpers S, Cyran CC, Schmidt M, Foerster S, et al. 18F-FDG PET/CT identifies patients at risk for future vascular events in an otherwise asymptomatic cohort with neoplastic disease. J Nucl Med. 2009;50:1611–20.View ArticlePubMedGoogle Scholar

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© Vancraeynest et al. 2016