Animals
Male, 7-week-old RccHan®: WIST rats (Wistar Hannover Rcc rats) were obtained from Japan SLC, Inc. (Shizuoka, Japan). They were allowed free access to tap water and fed a normal diet (CE-2; CLEA, Tokyo, Japan) or a choline-deficient, low-methionine high-fat diet (CDHFD; no choline, 45% fat, 0.1% methionine and 1% cholesterol) prepared by Oriental Yeast Co., ltd. (Tokyo, Japan), for 3–4 or 9–10 weeks. The rats were housed at a controlled temperature and under a 12-h light-dark cycle (lights on at 07:00 h). The experiments were approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine (approval number: 29-030-002, 21st July 2017).
Biochemical and histological analysis
After PET/CT scanning, all rats (5 animals/group) were euthanased by exsanguination under isoflurane anaesthesia. Plasma samples were collected and assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activity, and total cholesterol (TC), triglyceride (TG), glucose (GLU), total bile acid (TBA), albumin (ALB) and total bilirubin (T-BIL) concentrations by enzymatic methods using commercially available kits (Sekisui Medical, Tokyo, Japan) and a Hitachi 7170 autoanalyser (Hitachi, Tokyo, Japan), according to the manufacturer’s instructions.
Liver samples for protein analysis were quickly frozen in liquid nitrogen and stored at −80°C until use. The right hepatic lobes were fixed in 10% formalin, routinely processed, and embedded in paraffin. Four-micrometre-thick paraffin sections were prepared, and these were stained with haematoxylin and eosin (H&E) and Sirius red. Steatosis, inflammation, and ballooning were graded for severity on H&E-stained sections. Steatosis and inflammation were scored from 0 to 3: normal = 0; minimal = 1; moderate = 2 and marked = 3. Ballooning was scored from 0 to 2: normal = 0; minimal = 1 and marked = 2. NAFLD activity score (NAS) was then calculated as the sum of each of these scores. To assess hepatic fibrosis, Sirius red staining images were captured using a BZ-X700 microscope (Keyence Co., Osaka, Japan) and the Sirius red-positive area (%), corresponding to fibrosis, was measured using the BZ-X analysis application (Keyence Co.).
Protein analysis
Hepatic integrin αv and β3 subunit protein levels were determined using a JESS Automated Western Blotting system (ProteinSimple, San Jose, CA, USA). Liver tissue lysates were prepared in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific K.K., Tokyo, Japan), and lysates containing 0.2 mg/mL protein were separated using a 12–230 kDa Separation Module (ProteinSimple). Specific proteins were detected using mouse anti-integrin αv (1:50; BD Biosciences, San Jose, CA, USA; ab611012), rabbit anti-integrin β3 (1:50; Abcam, Toronto, ON, Canada; ab210515) and an Anti-Mouse Detection Module (ProteinSimple), according to the manufacturer’s instructions.
PET probe synthesis
18F-FPP-RGD2 was synthesized using a two-step method, as reported previously [20,23], to a specific activity of 273.3±60.7 GBq/μmol. The RGD dimeric peptide (PEG3-c[RGDyK]2) was purchased from Peptides International, Inc. (Louisville, KA, USA).
Analysis of the metabolites of 18F-FPP-RGD2 using thin-layer chromatography (TLC)
Metabolite analysis of plasma and liver samples from six normal diet-fed rats was conducted as described previously [24]. Briefly, ~20 MBq 18F-FPP-RGD2 was administered via a tail vein, then blood and liver samples were collected 30 and 90 min later under isoflurane anaesthesia. Blood was drawn from the abdominal vena cava, then the rats were exsanguinated, and their livers were collected and quickly homogenized on ice. Plasma was prepared by 1 min of centrifugation at 4°C and 20,817 × g. The plasma and liver samples were deproteinised by precipitation with acetonitrile, then centrifuged at 20,817 × g and 4°C for 5 min, and the supernatants were applied to RP-18 TLC plates (Merck KGaA, Darmstadt, Germany). The plates were developed at room temperature using 10% ammonium acetate/methanol (50:50) as the mobile phase, then dried and used to expose an imaging plate (Fuji Film Corp., Tokyo, Japan) for 30 min. With reference to the Rf value of an 18F-FPP-RGD2 standard, the distribution of radioactivity for 18F-FPP-RGD2 on the imaging plates was determined by digital PSL autoradiography using a Typhoon FLA 7000 imaging analyser (GE Healthcare, Uppsala, Sweden), and the data were analysed using Multi-Gauge imaging analysis software (Fuji Film Corp.).
Dynamic PET imaging
Dynamic PET/CT imaging was performed using a Triumph LabPET-12 PET/CT (TriFoil Imaging Inc., Chatsworth, CA, USA) and a PET camera with an intrinsic axial resolution of 1.38 mm FWHM (full width at half maximum) [25]. Under 2% isoflurane anaesthesia, a tail vein was catheterised for intravenous injection of the PET probe and the rats were placed on a heated pad on the scanner bed. Normal diet- and CDHFD-fed rats were imaged 3–4 or 9–10 weeks after starting the diets. Five rats per group were used for this experiment. At the start of the PET scan, 18F-FPP-RGD2 (15.5±2.2 MBq) was administered intravenously at a constant rate of 0.5 mL/30 s via a syringe pump (Legato 210; KD Scientific Inc., Holliston, MA, USA) in all animals. PET scanning was performed in dynamic scan mode for 90 min, then CT scanning was performed to acquire anatomical information and correct the PET images for attenuation.
For the analysis of the arterial input function (AIF) in the 6 normal diet-fed rats, a femoral artery was also catheterised (insertion length; about 5 cm from the femoral artery) for blood collection under 2% isoflurane anaesthesia and the cannula was flushed with heparinized saline. Arterial blood sampling was conducted 12 times over the following time periods: 0–10, 10–20, 20–30, 30–40, 40–50, 50–60, 90–100, 300–310, 900–910, 1,800–1,810, 3,600–3,610, and 5,390–5,400 s after 18F-FPP-RGD2 administration. Once blood sampling, the cannula was flushed with heparinized saline for the next sampling. The volume of blood removed during each time period was ~50 µL, making the total volume ~600 µL, which was ~5% of the total blood volume. Plasma was prepared by 1 min of centrifugation at 4°C and 20,817 × g. The radioactivity of the plasma was measured using a gamma counter (2480 Wizard2, PerkinElmer, Inc., Waltham, MA, USA), and was expressed as counts per minute/mL (cpm/mL). Each radioactive count was corrected for decay since the start of the gamma counting and was converted into an SUV.
Image processing and kinetic analysis
CT images were reconstructed using the filtered back-projection method (512 slices), and PET images were reconstructed into 25 frames of increasing length (6 × 10 s, 4 × 60 s, 11 × 300 s and 3 × 600 s) using the three-dimensional maximum-likelihood expectation maximization (3D-MLEM) algorithm and CT-based attenuation correction. To obtain time-activity curves (TACs) for kinetic analysis, the left ventricle (33 mm3) and liver (1,280 mm3) volumes of interest (VOIs) were manually defined for each animal on their CT images using PMOD PET data analysis software (v3.905, PMOD Technologies Ltd., Zurich, Switzerland). The VOI size was same in all animals. A spherical VOI on the left ventricle was used to obtain an image-derived plasma input function. TACs for the left ventricle and the liver were constructed by normalizing decay-corrected time-activity measurements to the injected doses of 18F-FPP-RGD2 and are expressed as mean standardized uptake values (SUVs), where SUV = radioactivity concentration (kBq/cm3) × body mass (g)/amount of radioactivity injected (MBq)/1,000.
Kinetic modelling to determine the total distribution volume (VT) of 18F-FPP-RGD2 was performed by fitting the TAC using a one-tissue, two-compartment (1T2C) and a two-tissue, three-compartment (2T3C) model using the PMOD software [26]. Figure 1 showed that (a) 1T2C and (b) 2T3C model in which, Cp, CT, CND and Cs represent the PET probe concentration of the plasma, total binding to integrin αvβ3, free or non-specific binding and specific binding to integrin αvβ3, respectively. K1, k2, k3 and k4 represent the extravasation rate of the PET probe, tissue efflux rate of free or non-specific binding probe, the rate of specific binding of PET probe to the extracellular portion of the integrin αvβ3 and dissociation rate, respectively. A physiological parameter of interest (VT; volume of distribution) was calculated for the tissue of interest. VT is a measure of the total (i.e. both non-displaceable and specific binding) distribution of the radioligand into the tissue and is equivalent to an equilibrium partition coefficient. VT was calculated by the following equation:
The Akaike information criterion (AIC) and curve fitting were used to determine the most appropriate compartment model for 18F-FPP-RGD2. AIC calculated by PMOD software was commonly used as statistic criteria [27] to compare the data fitting between different models [22,28,29], where lowest AIC value provides the most appropriate model. When we compared the VT calculated using the AIF and the VT calculated using the image-derived input function (IDIF), there is a stronger linear correlation between VT (AIF) and VT (IDIF) (Spearman's rank correlation rs = 0.943, P < 0.05) in the present study; therefore, IDIF was used in the present study. Models with irreversible binding were not considered because of the reversibility of the binding of this probe indicated by tissue TACs.
Statistical analysis
Comparisons of the two and four groups of rats were made using unpaired Student’s t-tests with Welch’s correction and Steel-Dwass test, respectively. Spearman's rank correlation was used to evaluate the relationships between two variables (integrin αv or β3 protein expression and SUV60–90 min, VT (IDIF), VT (IDIF) and VT (AIF)). Statistical analyses were performed using GraphPad Prism (version 6) statistical software (GraphPad Software, San Diego, CA) and SAS studio (version 3.71). P < 0.05 was considered to indicate statistical significance (two-tailed).