RESEARCH ARTICLE
Irbesartan Attenuates Atherosclerosis in Watanabe Heritable Hyperlipidemic Rabbits: Noninvasive Imaging of Inflammation by 18F-Fluorodeoxyglucose Positron Emission Tomography Yan Zhao, Keita Fukao, Songji Zhao, Ayahisa Watanabe, Tadateru Hamada, Kazuaki Yamasaki, Yoichi Shimizu, Naoki Kubo, Naoyuki Ukon, Toru Nakano, Nagara Tamaki, and Yuji Kuge
Abstract The purpose of this study was to assess the usefulness of
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F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in
evaluating the antiatherogenic effects of irbesartan, an angiotensin II type 1 receptor blocker. Watanabe heritable hyperlipidemic rabbits were divided into the irbesartan-treated group (75 mg/kg/d; n 5 14) and the control group (n 5 14). After a 9-month treatment, rabbits underwent 18F-FDG PET. Using the aortic lesions, autoradiography and histologic examinations were performed. PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant decrease in the 18F-FDG uptake level of irbesartan-treated rabbits (78.8% of controls; p , .05). Irbesartan treatment significantly reduced the plaque size (43.1% of controls) and intraplaque macrophage infiltration level (48.1% of controls). The 18F-FDG uptake level in plaques positively correlated with the plaque size (r 5 .65, p , .05) and macrophage infiltration level (r 5 .57, p , .05). Noninvasive imaging by
18
F-FDG PET is useful for
evaluating the therapeutic effects of irbesartan and reflects inflammation, a key factor involved in the therapeutic effects.
THEROSCLEROSIS is a chronic inflammatory disease in blood vessels that is related to the renin-angiotensin system (RAS).1 Through the angiotensin II type 1 (AT1) receptor, angiotensin II promotes atherosclerosis progression.2 Experimental studies and clinical trials demonstrated that treatment with AT1 receptor blockers (ARBs) can attenuate atherosclerotic plaque formation and reduce cytokine expression and inflammation levels.3 Positron emission tomography (PET) using 18F-fluorodeoxyglucose (18F-FDG) can noninvasively characterize the inflammatory activity within atherosclerotic plaques.4 The rationale for using 18F-FDG in imaging of vulnerable plaques has been demonstrated by numerous experimental reports,
A
From the Departments of Nuclear Medicine, Tracer Kinetics & Bioanalysis, and Integrated Molecular Imaging, Graduate School of Medicine, and Central Institute of Isotope Science, Hokkaido University, and Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Sapporo, Japan. Address reprint requests to: Yuji Kuge, PhD, Central Institute of Isotope Science, Hokkaido University, Kita 15 Nishi 7, Kita-ku, Sapporo 0608638, Japan; e-mail:
[email protected].
DOI 10.2310/7290.2015.00004 #
2015 Decker Intellectual Properties
and clinical applications also indicated the usefulness of 18FFDG PET for evaluating vascular diseases.5 Furthermore, the reduction in 18F-FDG uptake level in the aortic vasculature after therapeutic interventions such as lifestyle modifications or lipid-lowering therapies was also reported.4 Taken together, 18F-FDG PET should be useful for evaluating the therapeutic effects of ARBs because it reflects inflammation, a key factor involved in the therapeutic effects. However, such therapeutic effects of ARBs have not been evaluated by 18F-FDG PET, except for our preliminary study using 14C-FDG and autoradiographic methods in apolipoprotein E knockout mice treated with irbesartan.6 In this study, we treated a model of spontaneous atherosclerosis (Watanabe heritable hyperlipidemic [WHHL] rabbits) with irbesartan to demonstrate the usefulness of 18F-FDG PET in evaluating the antiatherogenic effects of irbesartan.
Methods Pharmaceuticals, Labeled Compounds, and Reagents Irbesartan was synthesized at Shionogi (Osaka, Japan). 18FFDG was obtained from Hokkaido University Hospital,
Molecular Imaging, 2015: pp 1–8
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which produces the tracer for clinical use. All other chemicals used were of the best grade available from commercial sources. Animal Studies Animal care and all experimental procedures were performed with the approval of the Animal Care Committee of Hokkaido University. Figure 1 shows the flow chart of the experimental design and animal treatment schedule. Studies were performed using male WHHL rabbits obtained from Kitayama Labs (Kyoto, Japan). Drug treatment was conducted at the animal facilities at Developmental Research Laboratories, Shionogi & Co., Ltd (Osaka, Japan). The rabbits were housed in individual cages under a 12-hour light/dark cycle (lights on 8:00 am to 8:00 pm), with controlled room temperature (20–26uC) and humidity (30– 70%). The rabbits were fed 100 g/d of a regular research diet (LRC4, Oriental Yeast Co., Ltd., Tokyo, Japan) and given tap
water ad libitum. At 3 months of age, the rabbits were weighed and divided into the irbesartan-treated and control groups. The irbesartan-treated rabbits (n 5 14) were fed with the LRC4 diet containing irbesartan (75 mg/kg/d).7 The control rabbits were fed the irbesartan-free LRC4 diet (n 5 14) throughout the experimental period. After 9 months of treatment, all animals were transported to Hokkaido University and maintained on an irbesartan-free LRC4 diet for 2 weeks before being subjected to PET study. Body weight and serum total cholesterol, blood glucose, blood urea nitrogen (BUN), and serum creatinine levels were measured before PET studies. PET Studies 18
F-FDG-PET imaging proceeded as previously described, with minor modifications.8 After a 4-hour fasting, the rabbits were anesthetized with 2.0 to 3.0% isoflurane inhalation and placed in a small animal PET scanner
Figure 1. Flow chart of the study design and animal treatment schedule.
FDG-PET Reflects Antiatherogenic Effect of ARB
(Inveon, Siemens Medical Solutions, Knoxville, TN) in the supine position. 18F-FDG was intravenously injected (192.9 6 10.0 MBq/rabbit), and a list mode acquisition was performed for each rabbit over 3 hours. The body temperature was maintained at 37.0 6 0.5uC during the PET scanning. The data from 150 to 180 minutes after 18F-FDG injection were reconstructed using the maximum a posteriori (MAP) algorithm without scatter and attenuation corrections. The image matrix was 256 3 256 3 159, resulting in a voxel size of 0.39 3 0.39 3 0.80 mm3. The locations of the aortic lumen and aortic wall were identified on the early blood-pool images (0–60 seconds after 18F-FDG injection). Two adjacent tubiform volumetric regions of interest (25 mm in length) were manually placed to include the vessel wall of the descending thoracic aorta for each rabbit. The 18F-FDG uptake level in the aortic wall was evaluated and calculated as the ratio to that in the liver.9 Four hours after 18F-FDG injection, the rabbits were sacrificed by exsanguination under deep pentobarbital anesthesia and their thoracic aortas were dissected into eight segments, embedded in Tissue-Tek medium (Sakura Finetechnical Co., Ltd.), and frozen in isopentane/dry ice. Serial cross sections of 10 mm (for autoradiographic exposure) or 5 mm (for histochemical staining) thickness were immediately cut and thaw-mounted on glass slides.
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Corp., Carpinteria, CA) using serial tissue sections to determine the macrophage infiltration level.8 Statistical Analysis Numerical parameters were expressed as mean 6 SD. Unpaired Student t-test was used to detect significant differences in each parameter between the control rabbits and the irbesartan-treated rabbits (Figure 2 and Figure 3). Linear regression analysis was performed to determine the correlation of 18F-FDG uptake level with histologic findings (plaque size and macrophage infiltration level) (Figure 4), and p , .05 was considered statistically significant.
Results Animal Characteristics and Serum Analysis Animal characteristics and serum analysis before the PET study are shown in Table 1. Compared to the control
Autoradiographic Studies The distribution of 18F-FDG in atherosclerotic plaques was determined by autoradiography, as described previously.8 Briefly, the cryostat cross sections from the thoracic aortas (eight segments for each animal) were exposed to phosphor imaging plates (Fuji Imaging Plate BAS-SR 2025, Fuji Photo Film Co., Ltd., Japan) together with a set of calibrated standards. 18F-FDG uptake levels (mean of eight segments) were calculated as percent injected dose (%ID) and normalized with animal body weight (%ID3kg). Coregistration of autoradiographic and histologic images was performed as described previously.10 Histochemical Studies Using the cryostat cross sections from the thoracic aortas, Movat’s pentachrome staining10 was performed to determine the total plaque size (mean of eight segments). Immunohistochemical staining was also performed using a rabbit macrophage-specific antibody (RAM-11, Dako
Figure 2. Representative 18F-FDG PET images (A) and quantification of 18F-FDG uptake levels in the thoracic aorta (B) of control and irbesartan-treated rabbits. 18F-FDG PET images clearly visualized the thoracic lesions of the control rabbit and showed a significant reduction in 18F–FDG uptake level in the lesions of irbesartan-treated rabbit. Black arrows highlight the thoracic aorta.
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Figure 3. Representative photomicrographs of Movat’s pentachrome staining and RAM-11 staining (A), autoradiographic (ARG) images (B), and their quantitative results. Irbesartan treatment significantly reduced plaque size, the intraplaque macrophage infiltration level (A), and the intraplaque 18F-FDG uptake level (B). Bar 5 500 mm.
rabbits, irbesartan-treated rabbits showed significantly lower total cholesterol and blood glucose levels. No significant differences were observed in body weight, BUN level, and serum creatinine level between the control rabbits and irbesartan-treated rabbits. PET Studies A higher 18F-FDG uptake level was found along the thoracic aortic wall in the control rabbits than in the irbesartan-treated rabbits (see Figure 2A, black arrows). Figure 2B shows the 18F-FDG uptake level in the aortic wall, which was significantly higher in the control rabbits than in the irbesartan-treated rabbits (1.23 6 0.29 versus 0.97 6 0.23; p , .01). Histochemical Studies Representative photomicrographs of specimens subjected to Movat’s pentachrome staining and RAM-11 staining are shown in Figure 3A. Plaques of the control rabbits showed
Figure 4. Correlation of the 18F-FDG uptake level with plaque size (A) and the intraplaque macrophage infiltration level (B). The 18F-FDG uptake level positively correlated with plaque size (r 5 .65, p , .05) and the intraplaque macrophage infiltration level (r 5 .57, p , .05).
significant intimal thickening with defused macrophage infiltration. In contrast, plaques of the irbesartan-treated rabbits only showed a thin intima with fewer macrophages. The plaques in the irbesartan-treated group were significantly smaller than those in the control group (control rabbits vs irbesartan-treated rabbits: 4.75 6 1.94 vs 2.05 6 1.54 mm2; p , .05). Irbesartan treatment significantly reduced the macrophage infiltration level (2.55 6 1.07 vs 1.23 6 0.91 mm2; p , .05). Autoradiographic Studies Representative autoradiographic images are shown in Figure 3B. The loci of elevated 18F-FDG uptake were consistent with the areas of high macrophage content (see Figure 3, A and B). Compared to the control rabbits, the 18 F-FDG uptake level was lower in the irbesartan-treated rabbits, which is in agreement with the macrophage infiltration. The 18F-FDG uptake levels in the plaques were significantly lower in the irbesartan-treated rabbits (74.61 6 38.27 vs 42.43 6 23.45 %ID3 kg 310–6; p , .05).
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FDG-PET Reflects Antiatherogenic Effect of ARB
Table 1. Animal Characteristics and Serum Analysis before the PET Study Parameter Body weight (kg) Total cholesterol level (mg/dL) Blood glucose level (mg/dL) Before the 4 hr fasting After the 4 hr fasting Plasma BUN level (mg/dL) Plasma creatinine level (mg/dL)
Control Rabbits (n 5 14)
Irbesartan-Treated Rabbits (n 5 14)
p Value
2.93 6 0.15 656 6 156
2.82 6 0.19 534 6 105
NS , .05
126 94 16.50 1.04
6 6 6 6
21 9 1.70 0.13
113 86 14.64 1.06
6 6 6 6
7 9 3.56 0.17
, .05 , .05 NS NS
BUN 5 blood urea nitrogen; NS 5 not significant; PET 5 positron emission tomography. Data represent mean 6 SD.
The relationships between the 18F-FDG uptake level and morphologic parameters are shown in Figure 4. The 18 F-FDG uptake level positively correlated with plaque size (r 5 .65, p , .05) and macrophage infiltration level (r 5 .57, p , .05).
Discussion In this study, we demonstrated the following: (1) in vivo 18 F-FDG PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant reduction in 18F-FDG uptake level in the lesions of irbesartan-treated rabbits; (2) the autoradiographic study also showed that the uptake level of 18F-FDG in plaque was significantly reduced by irbesartan treatment; and (3) histologic staining confirmed that irbesartan treatment can effectively suppress atherosclerosis progression and attenuate the intraplaque inflammation. To the best of our knowledge, the present study is the first to evaluate the antiatherogenic effects of ARBs by in vivo 18F-FDG PET imaging. As for monitoring the therapeutic effects of ARBs, the beneficial effects on plaque morphology have been confirmed by ultrasonography,11 cardiac computed tomography (CT), and magnetic resonance imaging (MRI).12 However, no previous study was reported on the use of nuclear imaging for evaluating the therapeutic effects of ARBs on atherosclerosis. 18 F-FDG PET imaging specifically evaluates functional changes in terms of the attenuation of intraplaque inflammation, which was found to be more critically involved in the therapeutic effect of ARBs, particularly for irbesartan. In addition, we previously confirmed that irbesartan halts the progression of atherosclerotic plaque formation by reducing the adhesion and infiltration of macrophages in apolipoprotein E knockout mice.6 This
therapeutic effect partially originates from the downregulation of inflammatory chemokines (e.g., MCP-1) through the blockade of the AT1 receptor, same as the other ARBs.13 Unlike most of the other ARBs, irbesartan activates peroxisome proliferator-activated receptor–c (PPARc).14 Because PPARc agonists can attenuate inflammatory gene expression by inhibiting proinflammatory transcription factors (e.g., nuclear factor kB),15 irbesartan can exert a further antiinflammatory effect.14 From the above-mentioned mechanism, evaluation of intraplaque inflammation status by 18F-FDG PET can directly reflect the antiinflammatory effect of irbesartan. Various components and molecular processes present in atherosclerotic plaques have been proposed as the target of the molecular imaging of vulnerable plaques.16 Superior to others, imaging of plaque inflammation has been demonstrated with clinical feasibility,5 and the utility of 18F-FDG PET has been noted.17 On the basis of this consideration, the application of 18F-FDG PET for monitoring the antiatherogenic effect of several drugs (e.g., statin, pioglitazone) was recently undertaken.18,19 ARBs are more widely used than statins or pioglitazone because hypertension is highly prevalent in the general population.20 Moreover, the blockade of the AT1 receptor by ARBs directly contributes to the suppression of inflammation in plaques.3 However, the therapeutic effects of ARBs on atherosclerosis had not been evaluated by 18F-FDG PET. Therefore, we preliminarily examined the 14C-FDG uptake level in the lesions of apolipoprotein E knockout mice with and without irbesartan treatment, and the results suggest the potential of 18F-FDG PET for evaluating the therapeutic effects.6 On the other hand, in another experiment, we observed delayed 18F-FDG elimination associated with ARB treatment,21 which may affect the 18FFDG uptake level in atherosclerotic lesions. In the same experiment, however, we demonstrated that a brief
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treatment cessation of ARB is useful for avoiding such effects and solving this problem. Accordingly, in the present study, irbesartan treatment was discontinued for 2 weeks in the irbesartan-treated group before being subjected to the PET study. Eventually, we demonstrated the usefulness of 18F-FDG PET for evaluating the antiinflammatory effects of irbesartan in atherosclerosis. On the other hand, the present experimental protocols prevented us from performing serial PET scans to track temporal changes in inflammation in plaques in the same animal, although such experiments would help strengthen the results of this study and be useful for assessing the efficacy of antiatherosclerotic drugs. For atherosclerosis research, several animal species and/ or animal models can be used. WHHL rabbits were used in this study because they have fewer species differences to humans in terms of lipid metabolism and atherosclerosis. In rabbits and humans, apolipoprotein B-100 is the major apolipoprotein of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). In contrast, in mice and rats, VLDL and LDL also contain apolipoprotein B-48, which results in the rapid clearance of these endogenous lipoproteins from the blood.22 Moreover, the cholesterol ester transfer protein (CETP) transfers cholesterol from high-density lipoprotein (HDL) to LDL and VLDL in rabbits and humans but not in mice and rats.23 WHHL rabbits show severe hypercholesterolemia owing to the genetic deficiency of the LDL receptor, and their atherosclerotic lesions morphologically resemble human plaques.24 However, the extremely high circulating cholesterol level in WHHL rabbits is not commonly observed in humans, and the aortic lesions of this model tend to be more inflammatory owing to the prolonged hypercholesterolemia.25 In this study, circulating cholesterol levels were high in both control and irbesartan-treated rabbits (see Table 1), and diffused macrophage infiltration was also observed in the plaques of control rabbits (see Figure 3A). Since 18F-FDG uptake is closely matched to the macrophage infiltration sites (see Figure 3B), intense 18F-FDG uptake along the vessel wall was clearly shown in PET images of control rabbits. However, it is reported that 18F-FDG uptake in the human aorta is predominantly regional and local to inflammatory active plaques.26 Thus, as the inflammatory status is certainly different between the two species, the effects of irbesartan on the 18F-FDG uptake levels in human plaque may not be as large as those shown in our WHHL rabbits. In addition, different from the high frequency of plaque rupture that occurs in humans, plaque rupture and occlusive thrombus are rarely observed in WHHL rabbits.27 Thus, careful interpretation is required to translate our
results into clinical settings. In this study, however, we showed a significant reduction in 18F-FDG uptake level in relation to the inflammation suppression in the lesions of irbesartan-treated rabbits (see Figure 3 and Figure 4). Our data showed that 18F-FDG PET can quantitatively reflect the changes in inflammation in plaques, which may validate the utility of 18F-FDG PET imaging in the evaluation of treatment efficacy, although further investigations, including on the sensitivity of 18F-FDG PET, are required in clinical settings. In addition to the antiinflammatory effect, irbesartan also exerts an antiapoptotic effect in atherosclerotic lesions.6 99m Tc-annexin A5 is one of the tracers that have been used in clinical settings as an apoptotic marker. The potential of 99m Tc-annexin A5 for evaluating the therapeutic effects of ARB on atherosclerosis was proved in our previous study.6 We also compared the localizations of 18F-FDG and 99mTcannexin A5 in experimental atheroma.28 99mTc-annexin A5 was more preferentially accumulated in unstable plaques than 18 F-FDG, although absolute accumulation levels were higher for 18F-FDG than for 99mTc-annexin A5. From the viewpoint of noninvasive in vivo imaging, however, the high uptake of 18 F-FDG may be more advantageous for the clinical evaluation of atherosclerosis. In addition, PET is superior for quantitatively evaluating tracer uptakes in plaques with high reproducibility.29 Furthermore, 18F-FDG PET has been widely applied in routine clinical diagnostic examinations for over three decades, whereas 99mTc-annexin A5 SPECT is still under investigation. Accordingly, in the present study, we used 18F-FDG PET and demonstrated its usefulness in evaluating the antiatherogenic effects of irbesartan. A limitation in our study should be considered. Our results only showed the preventive but not the regressive effect of irbesartan on atherosclerosis. In the present study, a long-term (9 months) and high-dose (75 mg/kg/d) irbesartan treatment was applied to young animals age 3 months, which show plaques only in the early stages. However, since the suppression of inflammatory infiltration critically contributes to the regression of atherosclerotic plaques,30 our results suggest the potential of 18 F-FDG PET for evaluating the antiatherogenic effect of irbesartan.
Conclusions We observed the reduction in plaque volume and the remission of inflammation in the atherosclerotic plaques of irbesartan-treated WHHL rabbits. Such effects of irbesartan on atherosclerosis can be noninvasively imaged by inflammation imaging by 18F-FDG PET.
FDG-PET Reflects Antiatherogenic Effect of ARB
Acknowledgments Financial disclosure of authors: This study was supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, Ministry of Education, Culture, Sports, Science and Technology, Japan. Prof. Yuji Kuge received a research grant from Shionogi & Co., Ltd. Drs. Toru Nakano, Ayahisa Watanabe, Tadateru Hamada, and Mr. Keita Fukao are current employees of Shionogi & Co., Ltd. The other authors have no relevant relationships to disclose. Financial disclosure of reviewers: None reported.
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