Study population
Patients who satisfied the following criteria were enrolled in the present study: 1) patients with hyperlipidemia [total cholesterol ≥220 mg/dl and/or low density lipoprotein (LDL) cholesterol ≥140 mg/dl, as defined by the Japan Atherosclerosis Society] or who needed lipid lowering therapy with statin based on the Japanese guidelines for the prevention of atherosclerotic disease (1–2 coronary risk factors: an LDL cholesterol ≤140 mg/dl; 3–4 coronary risk factors: an LDL cholesterol ≤120 mg/dl; or a history of ischemic heart disease despite an LDL cholesterol ≤100 mg/dl) and had no treatment with statins in the six months before enrollment, 2) patients with a high risk of atherosclerosis such as a severe plaque observed in the aorta or a carotid artery by past examination using CT or ultrasound, an aortic calcification observed through a chest x-ray, or metabolic syndrome, and 3) patients who required TEE due to atrial fibrillation or valvular heart disease. Patients were excluded if they had active infectious disease, poorly controlled diabetes, renal dysfunction or cancer found previously.
Study protocol
This was a prospective, randomized and comparative study called EPICENTRE study (Anti-inflammatory and Morphologic Effects of Pitavastatin on Carotid Arteries and Aorta Evaluated by Integrated Backscatter Trans-esophageal Ultrasound and PET/CT).
Patients were randomly divided into two groups: a pitavastatin treatment group (PI group: 2 mg/day), and a pravastatin treatment group (PR group: 10 mg/day). The dose of each statin (2 mg/day of pitavastatin and 10 mg/day of pravastatin) is intermediate dose in Japan. They each received treatment for six months, and the same examinations were performed after treatment at the same site in each patient. Blood biochemical examination was also conducted to assess lipid profile, glucose metabolism and the side effects of each medicine before and after treatment. This study protocol was approved by the ethics committee of our institution. Written informed consent was obtained from all enrolled patients.
PET/CT image acquisition and analysis
PET/CT images were obtained using methods as described in a previous study [5,7-10]. Patients were injected with 3.7 MBq /kg (0.1 mCi /kg) of 18 F-FDG intravenously. The injection was given after fasting for at least 8 hours. After one hour, two-dimensional whole-body PET/CT imaging was performed using a PET/CT scanner (Discovery ST Elite Performance, GE Medical Systems, Milwaukee, WI). This system can obtain PET and CT images (4.25-mm slice thickness) simultaneously. CT scan was performed for attenuation correction and PET images were reconstructed using iteration algorithm (128 × 128 pixel matrix). Images were obtained over 24 minutes. 18 F-FDG uptake was measured using a workstation (XTREX VIEW, J-MAC system, Japan).
At baseline, we manually found two regions that had a strong intensity of 18 F-FDG uptake in the thoracic aorta and carotid arteries in each patient and we defined them as target plaques. A region of interest (ROI) was placed on the target plaque as a spherical region 10 mm in diameter. We measured maximum SUV (max SUV) of the ROI and considered it as the max SUV of the target plaque. A target-to-background ratio (TBR) has recently been used as a method of normalization of the SUVs. It represents 18 F-FDG uptake by macrophages in inflamed vascular cells [4]. The TBR was calculated as the max SUV of target plaques divided by the mean SUV of blood which was measured in the right atrium. We were able to find target plaque easily after treatment using the distance from significant landmarks on PET/CT images (e.g., calcification of arterial wall, corpus vertebrae and bifurcations of an artery). The analyses of PET/CT were conducted by one radiologist and one cardiologist, who were blinded to the patients’ treatment assignment. The average of two measurements was used for the analysis. After six months later, we selected the same region as that selected at baseline by refereeing CT images.
Transesophageal echocardiography and conventional ultrasonography
TEE and conventional ultrasonography of carotid arteries were performed to measure intima-media thickness (IMT) and the corrected IBS (cIBS) values in the intima-media complex of the target plaques within two weeks after PET/CT. The IBS values in the intima-media complex were corrected by subtracting the IBS values in the tunica externa as follows [14]:
$$ \mathrm{cIBS}=\mathrm{I}\mathrm{B}\mathrm{S}\ \mathrm{values}\ \mathrm{in}\ \mathrm{the}\ \mathrm{aortic}\ \mathrm{wall} - \mathrm{I}\mathrm{B}\mathrm{S}\ \mathrm{values}\ \mathrm{in}\ \mathrm{the}\ \mathrm{tunica}\ \mathrm{externa}. $$
TEE was performed with an ultrasonic imaging system (SONOS 5500, Philips Medical Systems, Andover, MA, USA) and a 4–7 MHz multi-plane transducer with a 7.4 mm diameter “pediatric probe” (T6207, Philips Medical Systems, Andover, MA, USA) in the echocardiography laboratory by an operator who was blinded to the patients’ treatment assignment. The oropharynx was anesthetized with lidocaine before esophageal intubation. After the cardiac examination, the transducer was rotated in a posterior direction to obtain aortic images. The cIBS values in the intima and media of a target plaque and the intima-media thickness (IMT) at the same site were measured. The distance between the target plaque and the bifurcation of the left subclavian artery was measured by counting slices of CT, and target plaques were anatomically detected by TEE. If there was a characteristic calcification near the target plaque, it was also recorded for use as a reference point. The plaque was detected by this method with high reproducibility. Conventional ultrasound images and cIBS values of the carotid arteries were easily acquired at the same time using the same ultrasonic imaging system and a 3–11 MHz linear-array transducer.
We calculated average IMT and cIBS values of 5 slices spaced at-1 mm intervals around the target plaques. We detected two target plaques from thoracic aorta and two target plaques from right and left carotid arteries in each patient.
Measurement of c-IBS
IBS analysis was performed with a software package “Acoustic Densitometry” with the SONOS 5500. In this system, cIBS values are calculated as the average power of the ultrasonic backscattered signal from the region-of-interest (ROI) and represent the tissue characteristics. When measuring IBS values of plaques, ROIs were set on just the inner side of the tunica externa. The ROIs (21 × 21 pixels, 1.1 × 1.1 mm) placed at this site covered only intimal plaques.
The method of measuring IBS values was the same we previously reported [13]. We calculated averaged IMT and cIBS of 5 slices at 1-mm intervals around the target plaque.
Statistical analysis
Data are expressed as the mean ± one standard deviation or the number of patients (percentage). The normality of distribution was tested using the Kolmogorov-Smirnov test. The significance of the differences between groups that were normally distributed and had similar variances was tested by an unpaired Student’s t test. Otherwise, a Mann–Whitney U test was used to compare the difference between groups. Categorical data were summarized as percentages and compared using a Chi-square test or Fisher exact test. The relationships between the change of lipid profile and the change of TBR were tested for significance by linear regression analysis. All statistical analyses were performed using Stat View version 5.0 (SAS Institution Inc., Cary, NC, USA). A p-value < 0.05 was considered significant.