Cholesterol lowering depletes smooth muscle cell-derived fibromyocytes and chondromyocytes from atherosclerotic lesions
6 weeks old mice were treated with 5 daily injections (i.p.) 1 mg tamoxifen (T5648, MilliporeSigma) dissolved in corn oil to turn on TdTomato transgene in SMCs and allow lineage tracing of SMC-derived cells. Four weeks later, hypercholesterolemia was induced by an i.v. injection of rAAV8-D377Y-mPCSK9 virus particles (1 × 1011 vector genomes per mouse) followed by 16 weeks of High Fat diet (HF: S9167-E011, SNIFF Spezialdiäten GmbH, containing 25% fat with 8.9% coconut oil and 0.8% cholesterol) Mice are separated into two groups: treated with an high fat (HF) or a LDL-lowering diet (LF/ASO: low-fat diet combined with weekly injections (5 ug/g i.p.) of an antisense locked nucleic acid (LNA) oligonucleotide recognizing a sequence of apolipoprotein B (apoB) mRNA1). Mice were euthanized after 4 weeks or 12 weeks. The whole aortic arch (including ascending aorta, aortic arch and part of the descending thoracic aorta) was extracted and cleaned in situ for fat and surrounding tissues and pretreated with 2mg/ml collagenase II (Worthington LS004174) in Hanks' balanced salt solution (HBSS) for 15 min at 37ºC to easy removal of the adventitia. Stripped arches from each group (HF and LF/ASO) were washed in HBSS, open longitudinally and cut in small pieces and then placed in 3ml of enzymatic solution containing 200 µg/ml Hyaluronidase (Sigma-Aldrich, H3506); 250 µg/ml Liberase (Roche, 291963) and 200 µg/ml DNase (SigmaAldrich, DN25) for 60 minutes at 37ºC. Non-digested tissue was disaggregated using a cut Pasteur pipet with rounded edges. Reactions were stopped by adding PBS with 0.5%BSA. The suspension was passed through a 70 µm pore filter to remove aggregates. Cells were collected by centrifugation and resuspended in 300 µl PBS+0.5%BSA. DAPI at 1 µg/ml final concentration and Draq5 (Thermo Scientific 65-0880-96) at 5 µM final concentration, were added for dead/live cells detection. Viable cells, positive for Draq5 and negative for DAPI, were sorted using a FACSAria Cell Sorter. Sorted cells were recounted and their viability checked on a Countess III cell counter (Thermofisher). Up to 16500 cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics) for single cell isolation and cDNA reverse transcription and amplification. Sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) following the manufacturer instructions