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Basic Science Investigation |
1 Division of Cardiology, University of California, Irvine School of Medicine, Irvine, California; 2 Division of Cardiovascular Diseases, University of California, San Diego, La Jolla, California; 3 Department of Cardiology, Maastricht University Hospital, Maastricht, The Netherlands; 4 Division of Biochemistry, Maastricht University, Maastricht, The Netherlands; 5 Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 6 Armed Forces Institute of Pathology, Washington, District of Columbia; and 7 Department of Pathology, University of California, Irvine School of Medicine, Irvine, California
Correspondence: For correspondence or reprints contact: Artiom Petrov, PhD, Division of Cardiology, University of California, Irvine School of Medicine, Medical Science Building I, Room C 112, Irvine, CA 92697. E-mail: adpetrov{at}uci.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: apoE–/– mice • LDLR–/– mice • cholesterol diet • apoptosis • 99mTc-annexin A5
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Annexin A5 has been successfully used for noninvasive imaging of atherosclerotic lesions in an experimental rabbit model and preliminary studies of symptomatic carotid atherosclerosis in patients undergoing carotid endarterectomy (9,10). Annexin A5 is a naturally occurring protein and has a nanomolar affinity for binding to phosphatidylserine (PS), which is prominently expressed on the outer cell membrane surface of the apoptotic cells (11,12). Because apoptosis of macrophages contributes to the vulnerability of plaques to rupture (9), annexin uptake has been proposed to predict the likelihood of acute vascular events.
In the present study, we evaluated the ability of 99mTc-annexin A5 to detect the presence, prevalence, and magnitude of atherosclerotic lesions in the aorta of transgenic apoE–/– and LDLR–/– mice, to characterize the effects of a high fat/cholesterol diet on imaging parameters, and to assess whether the radiotracer uptake correlates with the presence of macrophages and apoptotic cells.
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MATERIALS AND METHODS |
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Annexin A5 and Radiolabeling
Human recombinant annexin A5, expressed in Escherichia coli, was derivatized with nicotinic acid analog hydrazinonicotinamide (HYNIC; Anor Med) by gentle mixing. HYNIC is a bifunctional molecule with an affinity for lysine residues of proteins on 1 moiety and for the conjugates of 99mTc on the other; the stable complex formed by this molecule did not affect protein bioreactivity (data not shown). To bind 99mTc to the HYNIC-annexin A5 conjugate, a reduced tin (stannous ion) and tricine solution (20 µL; 200 µL of 20 mmol/L tricine and 40 µg/mL Sn·2H2O) was added to 99mTc-pertechnetate (100 µL) with an aliquot of HYNIC-annexin A5 (100 µL) under anoxic conditions. The final specific radioactivity was 370–7,400 kBq/µg (10–200 µCi/µg) protein. Thin-layer chromatography showed a radiopurity of approximately 90%–98%.
In Vivo and Ex Vivo Annexin A5 Imaging Protocols
For atherosclerosis imaging targeting apoptotic cells, 99mTc-annexin A5 (175 ± 18 MBq) was injected intravenously through the dorsal tail vein and animals were anesthetized with 2.0% isoflurane for imaging procedures. For CT enhancement, a contrast agent (0.4 mL, Fenestra; Alerion Biomedical Inc.) was also injected through the jugular vein. Radionuclide imaging was performed 3 h after tracer injection using a dual-head micro-SPECT 
-camera with micro-CT (X-SPECT; Gamma Medica, Inc.). Micro-SPECT images of the aorta were acquired in 64 x 64 matrix, 32 stops at 120 s per stop on 140-keV photopeak of 99mTc with 15% windows using a low-energy, high-resolution pinhole collimator. The pinhole aperture size was 1 mm. Immediately after SPECT acquisition, a micro-CT scan was acquired without having to move the animals. The micro-CT used an x-ray tube operating at 50 kVp and 0.6 mA. Images were captured for 2.5 s per view for 256 views in 360° rotation. The micro-CT images were transferred to 256 x 256 matrix and micro-CT tomographic studies were fused, allowing the achievement of simultaneous scintigraphic and anatomic information in all tomographic scans in the 3 different spatial axes. After in vivo imaging, animals were sacrificed as detailed in the histology section, and aortas were carefully harvested after perfusion fixation. Planar images of the ex vivo aorta were acquired 5 h after tracer injection for 30 min in 128 x 128 matrix using a low-energy, high-resolution, parallel-hole collimator. A time-line diagram of the experimental protocol was shown in Figure 1.
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Quantitative Uptake of 99mTc-Annexin A5 in Atherosclerotic Lesions and Radiotracer Biodistribution
After ex vivo imaging, aortas were cut into 3 pieces (ascending aorta and arch, thoracic aorta, abdominal aorta). For quantitative uptake data, 99mTc uptake in the slices of the aorta was determined with -scintillation counting (1480 Wizard 3''; Wallac Co.) and expressed as the percentage injected dose per gram (%ID/g) aortic tissue. Similar biodistribution studies for the blood, heart, lung, spleen, and kidney were also undertaken and uptake was expressed as %ID/g.
Histologic and Immunohistochemical Evaluations of Atherosclerotic Lesions
Each specimen was subdivided into 5 or 6 equidistant sections and embedded in paraffin. Serial 5-µm-thick sections were cut and mounted on adhered slides pretreated with vectabond reagent (SP-1800; Vector). Deparaffinization was performed by heating the sections for 25 min at 56°C. The tissue was then dehydrated twice using a xylene bath and a graded series of ethanol. Tissue sections were stained with hematoxylin and eosin and Movat's pentachrome stain. Histologic specimens were analyzed on the basis of the classification scheme of the American Heart Association (AHA) (13).
For immunohistochemical characterization of cellular components of lesions in deparaffinized tissue sections, endogenous peroxidase was blocked by treatment with 3% H2O2 in PBS for 5 min at room temperature. Nonspecific background staining was blocked by a 1-h incubation in 3% bovine serum albumin with 0.3% Triton X-100 at room temperature. Macrophages were detected with Mac-3 antibody (0.16 µg/mL, 550292; BD Biosciences), and smooth muscle cells (SMCs) were stained with antiactin antibody (0.25 µg/mL, MAB1420; R&D). Sections were incubated with the primary antibodies overnight at 4°C and rinsed 3 times with PBS and 0.1% Triton X-100. The sections were then incubated with biotinylated secondary antibody followed by incubation with an ABC kit reagent (Vector) for 1 h each at room temperature. Finally, after washing 3 times with PBS, the sections were incubated for approximately 2–5 min with diaminobenzidine (DAB) (Vector) for the color reaction product.
In situ cell death (apoptosis) was detected by terminal deoxyribonucleotide transferase (TDT)-mediated nick-end labeling (TUNEL) staining, using, a TACS (Trevigen) apoptosis detection kit. Deparaffinized sections were treated with 3% H2O2 in doubly distilled water for 5 min to inactivate endogenous peroxidase. The sections were rinsed with PBS and microwaved for 2 min in preheated 0.01 mol/L citrate buffer using a 1.2-kW microwave oven at power level 4. Then sections were digested with 20 µg/mL of proteinase K in PBS for 15 min at room temperature. Exposed DNA fragments were labeled with biotinylated nucleotides and TDT overnight at 4°C. The incorporation of biotinylated nucleotides into DNA was detected with a streptavidin-conjugated horseradish peroxidase. A positive reaction was visualized with TACS blue label. Counterstaining with nuclear fast red was performed before final analysis of apoptosis cells.
Histochemical staining was observed under an Axiovert-200 inverted microscope (Carl Zeiss) and images were acquired with a Zeiss Axiocam high-resolution digital color camera (1,300 x 1,030 pixels) using Axiovision 3.1 software. Five to 8 images were acquired for each animal. These digital images were analyzed using KS300 analysis program (Zeiss). The percentage immunostaining area (immunostaining area/total image area x 100) was determined for all of the markers studied by averaging the percentage field area of several images per section that cover most or all of the region of study. Quantitative comparisons of the percentage immunostaining area with %ID/g uptake of 99mTc-annexin A5 were performed on sections processed at the same time.
Direct Localization of Annexin A5 in ApoE–/– Mice
In a separate experiment of 2 chow-fed apoE–/– mice, annexin A5 was localized in atherosclerotic plaques by histology. Biotinylated annexin A5 (0.5 mg, Apoptest-Biotin; NeXins Research) was injected intravenously. Thirty minutes after injection, the mice were euthanized and perfusion-fixed with 4% N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid–buffered formalin. The major arteries were carefully harvested and processed for paraffin embedding.
For annexin A5–biotin staining, serial sections (4 µm) were deparaffinized in xylene and treated with 0.3% H2O2 for 20 min to inactivate endogenous peroxides. Arterial sections were then incubated in a streptavidin-conjugated horseradish peroxidase overnight at 4°C. Annexin A5 was visualized with the chromogenic substrate DAB after enhancement with tyramide (catalyzed signal amplification system; DAKO). The apoptotic cells in the annexin uptake regions were identified with DNA fragmentation staining. The kidney was used as a positive control, as the biodistribution of annexin A5 is highest in this organ, and positive labeling represents successfully injected animals.
Statistical Analysis
The -scintillation counts were calculated as %ID/g of tissues or blood, and all data are presented as mean ± SD. To determine the statistical significance of differences between groups, 1-tailed ANOVA followed by the Scheffe post hoc test for multiple comparisons was done; P < 0.05 was considered as statistically significant. The extent of macrophage infiltration, SMC prevalence, and apoptotic cells were calculated as the percentage field area of several images per section that covered most or all of the tissue sections stained immunohistochemically. The data are also presented as mean ± SD for all groups of animals and statistical analyses were performed as described. The correlation between the radiolabeled annexin A5 uptake and the cellular composition was calculated by linear regression analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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The highest uptake of 99mTc-annexin A5 was in the kidney (104.7 ± 20.7 %ID/g), followed by liver (8.45 ± 2.46 %ID/g), spleen (4.96 ± 2.17 %ID/g), lung (2.36 ± 1.37 %ID/g), and heart (0.69 ± 1.06 %ID/g) in all animals irrespective of diet or genetic background. The annexin A5 uptake in blood was 0.30 ± 0.03 %ID/g, 0.32 ± 0.09 %ID/g, 0.43 ± 0.06 %ID/g, 0.47 ± 0.07 %ID/g, and 0.55 ± 0.07 %ID/g in control, chow-fed LDLR–/–, cholesterol-fed LDLR–/–, chow-fed apoE–/–, and cholesterol-fed apoE–/– mice, respectively.
Histologic Characterization of Atherosclerotic Lesions
Atherosclerotic lesions, ranging from foam cell–rich fatty streaks to fibrous plaques with large necrotic cores, were obtained. The necrotic cores comprised extracellular lipid, cholesterol clefts, and foam cells. Such plaques were typically observed in the aortic neointima of cholesterol-fed apoE–/–, chow-fed apoE–/–, and cholesterol-fed LDLR–/– mice (Fig. 4).
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Immunohistochemical staining confirmed the lesions to be rich in macrophage-derived foam cells, in some SMCs, and in a variable extent of macrophage apoptosis (Figs. 4B–4D). Macrophage infiltration was measured by Mac-3 surface antigens (Fig. 4B). Mac-3–positive cells were seen in significantly larger number in cholesterol-fed LDLR–/– (0.41% ± 0.25%) mice than chow-fed LDLR–/– (0.068% ± 0.011%) mice (P < 0.01) (Fig. 5A). On the other hand, macrophage contents were only marginally different in cholesterol-fed (0.52% ± 0.32%) mice versus chow-fed apoE–/– (0.42% ± 0.19%) mice (P = 0.59) (Fig. 5A). SMCs in the plaques were detected by 
-actin (Fig. 4C). SMCs prevalence was significantly lower in neointima as compared with macrophages (Figs. 4B and 4C). No significant difference was seen in -actin–positive cells observed in the fibrous cap of the plaques among the 4 transgenic animals (Figs. 4C and 5A). TUNEL staining was performed for the detection of apoptosis. Most apoptosis was found in the core region. The TUNEL-positive nuclei were more frequently observed in apoE–/– mice than in LDLR–/– mice, and more so in cholesterol-fed mice than in chow-fed mice (Fig. 4D).
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Direct Localization of Annexin A5 Uptake
The target for annexin A5 in atherosclerotic lesions was identified by administration of biotinylated annexin A5 in 2 chow-fed apoE–/– mice (Fig. 6). Thirty minutes after administration, the bulk of circulating annexin A5–biotin (Fig. 6B) was found to localize with macrophage-rich areas of the plaque in apoE–/– mice (Fig. 6C). A large number of cells showing annexin A5 uptake was found to be positive for apoptosis, as confirmed by the DNA fragmentation study (Fig. 6B, inset). Annexin uptake occurred in both apoptotic cells (black arrowhead) and nonapoptotic cells (black arrows) in macrophage-rich regions (Fig. 6B). Kidney sections from apoE–/– mice injection with annexin A5–biotin showed extensive staining in cortical regions (data not shown in Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Target for Annexin Imaging in Atherosclerotic Mouse Model
It is well established that annexin A5 binds to apoptotic cells with nanomolar affinity (11). Such cells express abundant PS on the cell membrane (10). Because apoptosis is strongly correlated with the vulnerability of plaque to rupture (14), annexin A5 imaging has been considered to be a surrogate for identification of unstable atherosclerotic lesions. The cell types undergoing apoptosis in the vessel wall are endothelial cells (15), vascular SMCs (16), inflammatory cells such as macrophages (14,17), and adventitial fibrosis (18). Apoptosis in vulnerable plaques is localized to inflammatory cells in the fibrous cap, deep intima, and shoulder regions; a fair degree of apoptosis is also found in macrophages surrounding the lipid core (17). It has been proposed that macrophage cell death may promote plaque instability by contributing to the size of the necrotic core (17). On the other hand, apoptosis of vascular SMCs within the fibrous cap may represent a chronic process leading to fibrous cap thinning (19). Extensive apoptosis of macrophages at the site of plaque rupture has been suggested to play a role in acute coronary events (14).
Noninvasive recognition of apoptotic cells has become possible by targeting of abnormal expression of PS on the cell membrane with the help of annexin A5. In previous rabbit and human studies, the binding of radiolabeled annexin A5 was increased in areas of atherosclerotic plaque and was predominantly localized to macrophages (9,20). These results are in concordance with our observation in apoE–/– and LDLR–/– mice. However, in the present study, radiolabeled tracer uptake was also seen in nonapoptotic macrophages. As inflammation constitutes the most important substrate for plaque vulnerability, annexin A5 imaging should be of immense value in the detection of unstable lesions. In clinical scenarios, PS is also expressed by crenating red blood cells in the regions of intraplaque hemorrhage that have also been proposed as the harbinger of plaque rupture (21). Therefore, molecular nuclear imaging may allow targeting of pathophysiologic process within the plaque critical to lesion instability.
Rosenfeld et al. reported that atherosclerotic lesions are distributed throughout the arterial tree of apoE–/– mice between ages of 24 and 60 wk (22). The innominate artery exhibits a highly consistent rate of lesion progression and develops vascular narrowing characterized by atrophic media and perivascular inflammation. The hyperlipidemic animals particularly demonstrate characteristics of advanced lesions in the innominate artery (22). Our results revealing maximal annexin A5 uptake in the aortic arch support the results of Rosenfeld et al. However, we used slightly older mice (mean age, 62 ± 0.9 wk old).
In a previous study using radiolabeled oxidation-specific antibody (oxidation-specific antibody to malondialdehyde-LDL [MDA2]) in LDLR–/– mice, enhanced 125I-MDA2 uptake correlated well with increased oxidized LDL (OxLDL) and macrophage immunostaining, suggesting plaque instability (23). In mice for which the high fat/cholesterol diet was withdrawn and replaced with normal mouse chow or chow supplemented with antioxidants, there was significant reduction in the 125I-MDA2 uptake, which was correlated with significant reduction in the macrophage content and OxLDL, detected by immunostaining with several murine and human OxLDL antibodies. Interestingly, in these areas of reduced 125I-MDA2 uptake, there was also increased SMC and collagen content, suggesting that the uptake of 125I-MDA2 reflected the development of plaque stabilization. This finding suggests that noninvasive imaging approaches may ultimately quantify and detect the presence of plaque stability or instability. Our results in the present study, which demonstrated that increased 99mTc-annexin A5 uptake was seen in plaques rich in macrophages and with a paucity of SMCs, are in accord with the previous results. Future studies can now be designed to longitudinally assess the regression of atherosclerosis in the same group of animals with noninvasive imaging models, such as with annexin A5.
Difference in 99mTc-Annexin A5 Uptake in ApoE–/– and LDLR–/– Mice With or Without Cholesterol Diet
In our results, the %ID/g 99mTc-annexin A5 uptake was higher in apoE–/– mice than in LDLR–/– mice regardless of diet. An earlier study had reported that the apoE–/– mice on a normal chow diet had much more profound hypercholesterolemia compared with the LDLR–/– mice (24). In addition, the animals exhibit quantitative and qualitative differences in plasma cholesterol levels. In the apoE–/– mice, cholesterol accumulated primarily in large lipoprotein particles, primarily in intermediate-density lipoproteins, but also in very-low-density lipoproteins and chylomicron remnants (1,2). In contrast, the LDLR–/– mice had a much more selective elevation in LDL (3,8). We did not measure the plasma levels of cholesterol in this study.
In the present study, the %ID/g 99mTc-annexin uptake was higher in cholesterol-fed transgenic mice compared with chow-fed mice. As previously reported, an abundant cell-poor, lipid-rich core developed in the atherosclerotic lesions of cholesterol-fed animals (25). The core was characterized by extracellular lipid deposition, cholesterol crystal, and cell death and was situated in the deep layer of the plaque. In experimental studies using hyperlipidemic mice, apoptosis was also observed in the advanced vascular lesions of APOE*3-Leiden transgenic mice (26) and the aortas of hyperlipidemic apoE–/–- and LDLR–/–-deficient mice (27). Apoptosis was involved in the active turnover of foam cells of both macrophage and SMC lineage, even in the early atherosclerotic lesions of transgenic mice. However, the effects of additional cholesterol on atherosclerotic lesions remain to be investigated. Our results, which demonstrated that additional cholesterol diet for hyperlipidemic transgenic mice increases the presence and extent of apoptosis, may provide an important standpoint with regard to plaque stabilization rather than plaque regression for advanced atherosclerotic lesions.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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