All chemicals used in these studies were reagent grade. FDG (F-5006) and FDG-6-P (F-6037) were from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 was from Hyclone (Logan, UT, USA) and 10% FBS was from Invitrogen (Carlsbad, CA, USA). ProSense750 was from VisEn (Woburn, MA, USA).
THP-1 cells were obtained from ATCC. The cells were cultured in RPMI 1640+10% FBS +1% anti-anti (Invitrogen) at 37°C, 5% CO2. Cells were harvested non-enzymatically with enzyme free cell dissociation solution (Specialty Media) and resuspended in RPMI 1640 without glucose. Cell concentration was determined using hemocytometer with trypan blue exclusion. Cells were plated in triplicate in a 96 well plate at different concentrations (1, 0.5, or 0.25×106 cells/ml) in 200 µl RPMI +10% FBS +5.5 mM FDG and incubated for 2 hours at 37°C, 5% CO2. After incubation, the media was removed, and the cells were lysed in 150 µl MeOH/CH3CN/formic acid. The organic lysates were stored covered at 4°C until assayed for FDG-6-P by mass spectrometry. An identical set of wells was prepared and used to measure protein concentration; at the end of the incubation cells were lysed in 50 µl radioimmunoprecipitation assay buffer and protein concentration determined using a BCA protein assay kit (ThermoScientific). Protein concentration was used to normalize FDG-6-P data.
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Experimental procedures using animals were approved by the Eli Lilly Institutional Animal Care and Use Committee. ApoE KO and C57Bl6J mice were from Taconic (Hudson, NY, USA).
To evaluate FDG-6-P in spontaneous atherosclerotic lesions, one group of ApoE KO mice (n
6) was fed standard chow for 3 months, while another group (n
5) was fed Western diet containing 0.21% cholesterol and 21% fat for the same period of time. Age-matched C57BL6/J mice (n
6) were fed normal chow and served as a control.
To accelerate lesion formation, 7-week old ApoE KO mice were pre-fed with the Western diet for 14 days, and then their left common carotid arteries were ligated under isofluorane anesthesia as described previously 
. Mice were kept on the same diet following the ligation surgery and were euthanized 14 days after ligation by carbon dioxide asphyxiation. 10–12 animals per group were utilized for various experimental protocols, including the drug treatment.
To optimize the dose of the compound, T0901317 was administered daily at doses of 0, 3, 10, or 30 mg/kg of weight by oral gavage (10 ml/kg). Vehicle used was 1% hydroxyethylcellulose and 0.25% Tween80 and was similarly administered to control animals. A compound was administered for two weeks, starting at the day of ligation. After obtaining the dose response data, all follow-up experiments have been done with the 10 mg/kg dose of T0901317. This dose was used to evaluate various analytical endpoints described below.
Near-infrared fluorescence (NIRF) imaging
To obtain an independent measurement of macrophage activation in accelerated atherosclerotic lesions, the carotid arteries from ApoE KO mice on Western diet with carotid ligation were imaged ex vivo with the imaging probe ProSense750. ProSense750is activated by proteases, including Cathepsin B, L and S, and transitions from a non-fluorescent to fluorescent form upon activation. Compound treatment experiments were conducted as described above. 24 hours before euthanasia, ProSense750 (2 nmol in 150 µL of PBS) was administered via the tail vein. On the day of imaging, the animals were perfused with saline via the left ventricle. Ligated and unligated (contra-lateral) common carotid arteries were dissected, cleaned and imaged. NIRF reflectance was measured using the Maestro multi-spectral instrument (CRi (now PerkinElmer), Hopkinton, MA) deep red filter set (750 nm lp) with a 3000 ms exposure and 2×2 binning. Data was analyzed using the Maestro Software 2.4.2 and ImageJ.
Tissue cholesterol analysis
Dissected carotid arteries were immersed in 2
1 chloroform/methanol, and lipids were extracted overnight. Free cholesterol and cholesterol esters were analyzed by LC/MS/MS as described previously 
. Delipidated tissues were either embedded into paraffin for further histological analysis or re-extracted for FDG-6-P analysis (vide infra).
For histology, 10 equally spaced (200 µm) paraffin cross sections were stained using modified Masson's trichrome procedure that included elastin staining. Macrophages were visualized immunohistochemically using MAC-2 (DAKO). Trichrome-stained sections spanning the entire length of the artery (3 per artery) were used for morphometric analysis. Lesion area was calculated using Image-Pro Plus Version 5.0.1. The lesion area was defined as the region between the lumen and the internal elastic lamina.
FDG was dissolved in saline to a final concentration of 10 mg/ml. FDG solution was administered at the dose of 30 mg/kg via tail vein injection 30 min before euthanasia. Selection of dose and timing is based on the preliminary experiments (data not shown). After the FDG injection, animals had access to food and water ad libitum. As described above, the mice were euthanized by carbon dioxide asphyxiation. The animals were perfused with saline via the left ventricle. After perfusion, aortas from the mice with spontaneous atherosclerosis as well as ligated and unligated (contralateral) common carotid arteries from the mice with accelerated atherosclerosis were dissected. The arteries were placed in chloroform:methanol (2
1) overnight for lipid extraction. Other tissues (e.g. spleen) were frozen on dry ice.
Preliminary studies were conducted by spiking authentic FDG-6-P into biological matrices followed by extraction with various solvents. The method entails extraction of tissue samples with 4X (by weight) volumes of 50% acetonitrile-50% methanol-0.1% formic acid. Tissues were extracted with sonication. Homogenate was centrifuged and clear supernatants transferred to vials or microtiter plate for LC/MS/MS analysis (using 5–10 µl of extract). In the case of mouse carotid artery (dry weight <10 µg) we used 150 µl of extraction solvent to prepare analytical samples.
FDG-6-P mass spectrometry
The analysis of 2-FDG-6-P was carried out using an Agilent 6140 series triple quad LC/MS/MS with MassHunter data analysis software (Agilent Technologies, Inc., Santa Clara, CA, USA) fitted with an electrospray ion source and run in negative ion mode. Detection was accomplished by monitoring the precursor ion of 2-FDG-6-P with mass-to-charge ratio (m/z) of 261 and targeting its product ion with m/z set to 139.2. The chromatographic separation employed a Cogent Diamond Hydride HPLC column, 4 µm, 100 A, 100 mm×2.1 mm ID (from MicroSolv Technology Corp., Eatontown, NJ, USA) and a mobile phase consisting of 3% acetonitrile in water with an overall 0.1% formic acid content with a flow rate of 0.75 ml/minute. Clearly delineated chromatographic peaks with the retention time of authentic standards and expected molecular mass were seen after each injection of sample. Analytes were quantified based on the area of these peaks.
All values are expressed as mean ± SEM. Dose response and time course data were analyzed using one-way ANOVA. Comparisons of the left (ligated) and right (unaffected) carotid arteries in a mouse model of accelerated atherosclerosis were made by a two-tailed, paired t-test, because pairs of plaque-covered and plaque-free arteries were always obtained from the same animal. Comparisons of ligated arteries from different animals were assessed using an unpaired t-test.