Regulation of glycolytic activity in multiple cell types explains the ability of FDG-PET to measure disease activity in atherosclerosis
Regulation of glycolytic activity in multiple cell types explains the ability of FDG-PET to measure disease activity in atherosclerosis
Animals were euthanized and the abdominal aorta and iliac arteries were quickly isolated and opened. The atherosclerotic plaque was dissected from the distal 0.5 cm and proximal 0.5 cm of the right iliac artery. The media, adventitia, and perivascular tissue were manually removed and approximately 200-250 mg of plaque was selected, washed in HBSS, and minced. It was incubated for 60 minutes at 37 °C in 1 ml of an enzyme solution containing 1,5 mg/ml (8 U/ml) of elastase (LS002279, Worthington Biochemical), 2 mg/ml of liberase (05401119001, Sigma Aldrich) and 300 µg/ml of DNaseI (Sigma Aldrich, DN25). Non-digested tissue was disaggregated using a smooth-tipped Pasteur pipette. Reactions were stopped by adding PBS containing 0.5% bovine serum albumin (BSA). The suspension was passed through a 70 µm pore filter to remove aggregates. Cells were collected by centrifugation and  resuspended in 500 µl PBS+0.5% BSA. DAPI (final concentration, 1 µg/ml final ) and Draq5 (Thermo Scientific 65-0880-96; final concentration, 5 µM) were added to enable detection of viable and dead cells. Viable cells (Draq5 positive and DAPI negative) were sorted with a FACSAria Cell Sorter.  BMS-212122 is a chemical inhibitor for Microsomal Triglyceride Transfer Protein (MTP) with the IUAPC name 9- {4- [2,5-Dimethyl- 4- ({[4'-(trifluoromethyl)-2-biphenylyl] carbonyl} amino)-1H-benzimidazol-1-yl] butyl}-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide.Atherosclerosis was induced in PCSK9D374Y transgenic minipigs (males) by feeding a standard pig feed supplemented with 20% w/w lard, 1.5% w/w cholesterol (HFHC), 0.7% NaCl w/w, from 3 until 15 months of age. During the first 6 months, the diet was also supplemented with 0.7% cholic acid. Animals were then either continued on the HFHC diet for 3 months (HFHC group) or changed to the standard pig feed with supplemented BMS-212122 (LF/MTPi group). The BMS-212122 dose was 1 mg/(kg·day)  during the first week and 0.5 mg/(kg·day) for the rest of intervention period. Up to 16500  cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics). After reverse transcription and cDNA amplification, sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).