Conceived and designed the experiments: YYY GJT. Performed the experiments: YYY YYW YZ ZLS SW FF. Analyzed the data: YYY YYW YFL. Contributed reagents/materials/analysis tools: GSM NFL. Wrote the paper: YYY.
The authors have declared that no competing interests exist.
Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker.
Twenty-six male apoE−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence.
SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.
Abdominal aortic aneurysm (AAA) is a major cause of mortality in the elderly population due to an increased risk of rupture
AAA is characterized by tissue degeneration, infiltration of inflammatory cells and subsequent dilation of the vessel
High-resolution MRI has emerged as the leading non-invasive
The aim of the present study was to perform an evaluation of SPIO as an
We followed the experimental study design as shown in
Saline or ANG II were administrated
Control | Saline | ANGII 500 ng/kg/min | ANG II 1000 ng/kg/min | |
Body weight (g) | 26.5±2.4 | 29.9±2.6 | 28.0±2.3 | 28.0±1.2 |
Mean blood Pressure (mmHg) | 88.8±7.7 | 94.3±15 | 110.9±11 | 120.8±6.3 |
Cholesterol (mg/dl) | 103.9±35 | 696.7±114 | 641.3±114 | 656.4±91 |
Triglycerides ( mg/dl) | 53.2±4.2 | 122.9±24.9 | 107.8±20.8 |
114.8±14.4 |
MCP-1 (pg/ml) | 16.6±7.2 | 32.4±6.2 | 64.8±9.2 |
84±14 |
Lumen diameter (mm) | 0.98±0.1 | 0.92±0.2 | 1.38±0.1 |
1.82±0.2 |
Data are presented as mean± SEM.
In apoE−/− mice infused with ANG II (500 or 1,000 ng/kg/min), the mean arterial pressure was elevated within 14 days of infusion, but no significant effects on body weight, total serum cholesterol, or triglycerides concentrations were observed (
MCP-1, a macrophage chemotactic factor, was examined in serum samples obtained from all animals at the end of study. There was a significant elevation in MCP-1 in the 500 or 1,000 ng ANG II groups (64.8±9.2 or 84±14 pg/mL) compared to the saline group (32.4±6.2 pg/mL,
Aneurysms were clearly visualized on MRI in both groups of ANG II treated mice. Dilation of the abdominal aorta was identified in the suprarenal abdomen in these mice. None of the saline-treated apoE−/− mice developed an aneurysm. The aneurysm was seen as a region of inhomogeneous low signal intensity compared with the bright signal in the aortic wall on T2-weighted (T2WI) MRI. Narrowing of the vessel lumen due to a large volume of mural thrombus was also noted (
(A) Representative abdominal aorta tree images in mice acquired at 7 T using 3D-FLASH sequence. The longitudinal view shows the characteristic dilatation of the abdominal aorta (B)
On 3D FLASH MRI images, the longitudinal view showed an example of the characteristic dilatation of the abdominal aorta in the suprarenal region in ANG II treated mice (
(A) The abdominal aorta before SPIO contrast agent administration and the same segment of the abdominal aorta 24 h post-SPIO administration. Red arrows indicate aortic aneurysms. (B) MRI signal in the abdominal aorta of apoE−/− mice after injection of contrast agents. Values are expressed as mean ± SEM (n = 6, *P<0.05 vs. Previous & ANG II 14D).
Macrophages were observed entering the aneurysm from the shoulder and adventitial areas in the ANG II–induced mice (
(A) Upper panel: immunohistochemical analyses of aortic aneurysms, lesion area stained positive for Mac-3 (200×). Lower panel: the corresponding area of Prussian-blue staining demonstrated localization of iron positive staining (200×). (B) Double staining of CD68 positive macrophages (brown) and iron particles (blue) was performed. Upper panel is the 500 ng ANGII group, and lower panel is the 1000 ng ANGII group. Double staining confirmed that the high dose ANGII caused macrophage accumulation. (C). Quantitative analyses of Mac-3-positive cells. (D). Double staining of α-SMA positive smooth muscle cell (brown) and iron particles (blue) was performed. Red arrow indicated Prussian-blue positive cell, black arrow indicated SMC. SMC/Prussian-blue staining did not exhibit colocalization. (E). In the 1000 ng/kg/min ANG II-induced group without SPIO injection, Prussian blue-stained hemosiderin (blue) is shown in macrophages (brown) of the inner adventitia (left). Corresponding section of the SPIO injection group, with numerous Prussian blue-stained macrophages in the adventitia (right). (F). ANG II induced MMP activity. Representative images of MMP-9 immunostaining in the AAA area (200×).
Immunohistochemistry revealed a marked upregulation of MMP-9 expression in the suprarenal aorta of ANG II-induced mice as compared to the aortic tissue of mice infused with saline (As shown in
Compared with measurements at baseline before ANG II infusion (34.1±6.5, n = 11), MRI signals at aneurysm areas were significantly decreased in the ANG II-induced groups (18.7±4.1, n = 6,
Animal care and experimental protocols were approved by the Southeast University Committee on Animal Care (approval ID: SYXK-2009.2981). Thirty-six week old apoE−/− male mice, and background strain C57BL/6J mice, were provided by the Animal Center of Peking University Health Science Center (Beijing, China). Mice were fed with a standard lab chow. Mice were housed individually and were allowed free access to chow.
Animals were assigned to four groups: saline group received a saline infusion (n = 6), the 500 ng group received an ANG II infusion delivered at 500 ng/kg/min (n = 10), the 1000 ng group at 1000 ng/kg/min (n = 10), and C57BL/6 mice were used as the control group in the study (n = 5). ANG II (Sigma, St Louis, MO, USA) infusions were administered subcutaneously using osmotic mini pumps (Alzet, Model 1002) for 14 days at either 500 ng/kg/min (500 ng group) or 1000 ng/kg/min (1000 ng group). Mice were anesthetized with isoflurane gas during implantation and removal of osmotic mini pumps. Mini pumps were implanted subcutaneously in the mid-scapular region of the mice at day 1, and removed on day 14.
Fourteen days following ANG II or saline treatment, all animals underwent MRI scanning and administration of Resovist (1 mmol/kg iron, diluted with saline, three minutes)
MRI scanning was performed at baseline prior to ANG II or saline administration, and at the 14th day following ANG II infusion and days 1 and 2 after administration of SPIO.
All scanning was performed on a micro-MR animal scanner (7.0T Bruker PharmaScans, Bruker Biospin, Ettlingen, Germany). Each mouse was induced and maintained under isoflurane anesthesia (2%) in medical-grade air and monitored using the small animal instrument monitoring and gating system for respiration rate (reduced respiratory rate to 40 breath/min). All animals were placed in the supine position.
For MRI angiography, the abdominal aortic trees were imaged using a three-dimensional fast low-angle shot (3D-FLASH) sequence (repetition time (TR) was 15 ms, echo time (TE) was 2.5 ms. The field of view (FOV) was 3.5 cm×3.5 cm, and a 256×256×128 matrix was employed, yielding an voxel resolution 137×137×156 µm; flip angle (FA) was 20°; 1 excitations).
The suprarenal abdominal aorta was identified on the scout view of the coronal images. Eighteen contiguous, 1000-µm thick axial slices spanning from the abdominal aorta between the diaphragm and the renal artery were acquired using a spin echo sequence. MRI images were obtained using black-blood T2 to proton density (PD)-weighted multi-spin multi-echo (MSME) sequence and a dedicated mouse volume coil. Imaging parameters were as follows: TR 1206.9 ms, TE 12.8/34.2 ms, FOV 2.5 cm, FA was 180°, matrix size 256×256, and in-plane resolution 141×141×800 µm3; slice thickness of 1 mm, and four excitations. The imaging acquisition time was 30 minutes per animal. Fat suppression was performed for proton density and T2-weighted imaging. Coronal T1-weighted (MSME; TE 15 ms, TR 664 ms, 1 mm slice thickness, matrix 256×256, field of view (FOV) 2.5 cm, eight averages, 18 coronal slices, scan time 25 minutes) was acquired at exactly the same position.
Images were analyzed using the ParaVision software (PV5.0, Bruker, Germany). Continuous imaging on slices (n>5) allowed us to follow the CNR in the abdominal aorta over time. Signal intensities were measured by manually drawing a region of interest (ROI) within the wall of the suprarenal abdominal aorta and the vessel lumen. CNR was calculated for the signal intensity changes in similar sized regions of interest placed in the aortic lumen as well as within the vessel wall using the following equation: CNR = (wall signal−blood signal)/(standard deviation of the muscle signal).
Following MRI acquisition on day 16, blood pressure was measured as previously described
After blood collection, lethal doses of pentobarbital (120 mg/kg, IP) were administered. The collected blood was centrifuged at 1500 rpm for 10 minutes, and serum was separated and collected. Total cholesterol and triglyceride levels were assayed using diagnostic kits.
Monocyte chemotactic protein-1 (MCP-1) was measured in serum using an ELISA kit (Pierce Chemical Co. Rockford, IL).
At the end of the procedure, the abdominal aorta was fixed in 4% paraformaldehyde and embedded. Serial sections of the abdominal aorta were cut at 3-mm intervals matching corresponding MR images. Transverse sections in 5 µm thickness were cut and stained with hematoxylin-eosin (HE). Each image was digitized with a camera and analyzed using a microscope. Prussian blue and MAC-3 (BD Biosciences Pharmingen, San Diego, CA), stains were used for detection of SPIO particles and macrophages, respectively. Sections were subjected to immunohistological analyses of matrix metalloproteinase 9 (MMP-9, BD Biosciences). To verify SPIO uptake by macrophages, serial sections were dually stained for Prussian blue iron and MAC-3. At least 10 sections were stained per mouse and quantification was performed blindly.
For CD68 immunohistochemical analyses, the abdominal aortas were collected from sacrificed mice, immediately placed in embedding medium (OCT compound), and rapidly frozen using liquid nitrogen. Frozen sections were prepared on a Leica CM1950 Cryostat (Leica Instruments, Heidelberger, Germany). Rat Anti-Mouse CD68, a tissue macrophage marker (clone FA-11; BioLegend, San Diego, USA), was used (1∶100) to access the deposition of macrophages in the abdominal aorta. For colocalization of SPIO particles, transverse sections were subjected to immunohistological analysis of macrophages (CD68), smooth muscle cells (rabbit against α-SMC, Abcam, Cambridge, UK), SPIO uptake was evaluated by Prussian blue staining on the same section.
Data was compared among experimental groups using ANOVA followed by Fisher's protected least-significant differences (PLSD). Data was expressed as mean ± standard error of the mean (SEM). Differences were considered statistically significant at a value of P<0.05. A Pearson correlation coefficient analysis was calculated to describe the relationship between MR and histological measurements.
This study demonstrates that the non-invasive assessment of SPIO contrast agent uptake
MRI can provide anatomical, structural and functional characterization of the arterial wall
Interestingly, the presence of acute thrombus formation was observed in one mouse in the 1000 ng group, as shown in
Compared with 14 days of ANG II infusion, MRI at day 15 showed acute artery occlusive and mural thrombi (red arrow hyperintense on T1-weighted images). HE showed fresh thrombus with platelets, inflammatory cells, and fibrin mesh.
Research has demonstrated that assessment of USPIO uptake with MRI can be used to detect focal hotspots of inflammation in asymptomatic AAA
AAA almost always relates to atherosclerosis, which is essentially a chronic inflammatory process, but the pathophysiology of AAA is quite different from that of non-aneurysmal atherosclerotic plaques. In AAA, inflammation is mostly confined to the media and adventitia of the aorta, whereas in atherosclerotic plaques, the inflammatory reaction is seen primarily in the intima
Resovist
It was observed that the SPIO-laden macrophages, which are abundant in the adventitial of the ANG II infusion groups, were minimal in atherosclerotic plaques in the saline-infusion group. Therefore, SPIO appears not suitable for the detection of plaques presumably because of its relatively large size which results in reduced transendothelial passage, tissue penetration, and easy uptake by the RES
In a recently published article, SPIO-enhanced MRI visualizes leukocyte phagocytic activities in AAA patients
To determine the origin of iron in Prussian-blue-positive macrophages, we further analyzed the SPIO-laden macrophages dependent on two points. We found that the number of Prussian blue-positive cells was much less in the mice without SPIO injection compared to the ones with SPIO injections. Therefore, imaging of iron phagocytic activity could be enhanced using intravenous injection of SPIO. Furthermore, the locations of hemosiderin-laden macrophages in non-SPIO injection group are associated with hemorrhages in the inner adventitia (
This study focused on early inflammatory events in an ANG II-induced AAA in a mouse model. Following 14 days of ANG II infusion, activated macrophages released cytokines and proteolytic enzymes. MMP-9 has also been found in the aneurysm vessel wall which primarily colocalized with infiltrated macrophages. Since AAA progression and eventual aortic rupture depends on the activity of macrophage-derived MMP-9
In summary, this study demonstrates that imaging of macrophage activity in the vessel wall with SPIO provides a valuable tool for studying aneurysm biology. But signal loss on T2WI in the aneurysm vessel wall is also partially influenced by endogenous hemosiderin iron from intramural hemorrhages and thrombi. Noninvasinve visualization of SPIO-laden macrophages within an aneurysm may provide physiological information other than size in assessing the risk of acute AAA rupture.
We thank Dr. Yufei Xue and Dr. Weiping Wang for their valuable contributions in the manuscript revision.