S00725

Human aortic smooth muscle cells (HAoSMC) were purchased (C0075C, Gibco™, Thermo Fisher) and maintained with human VSMC basal medium (M231500, Gibco™, Thermo Fisher) with smooth muscle growth supplement (SMGS) (S00725, Gibco™, Thermo Fisher). Human aortic endothelial cells (HAoEC) were purchased (C12271, PromoCell, Heidelberg, Germany) and maintained with endothelial cell growth medium (C-22022 and C-39226, PromoCell). Human umbilical vein endothelial cell (HUVEC) was purchased (CC-2519, Lonza) and maintained with EGM™-2 Endothelial Cell Growth Medium-2 Bullet Kit (CC-3162, Lonza). THP1 was purchased (40202, Korean cell line bank) and maintained with RPMI 1640 medium (11875093, Gibco), 10% heat inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Gibco, Carlsbad, CA). Cells were cultured at 37 ℃, at humidified 5% CO₂ incubator unless mentioned otherwise. When reached confluence, the cells were sub-cultured using StemPro Accutase (A11105-01, Gibco). Culture media were changed every 3 days and passage numbers from 5 to 9 for primary cells were used for the experiments.

We purchased pooled human umbilical vein endothelial cells (HUVEC; C2519A), human aortic endothelial cells (HAEC; CC-2535), and human coronary artery endothelial cells (HCAEC; CC-2585; Lonza) and cultured them in EBM-2 media supplemented with EGM-2 or EGM2-MV SingleQuot kit. We purchased immortalized HAEC (teloHAEC; CRL-4052; ATCC) and cultured them in vascular cell basal medium supplemented with vascular endothelial cell growth kit and puromycin at 0.3 μg/mL. Human coronary artery smooth muscle cells (HCASMC) and human aortic smooth muscle cells (HASMC) were obtained from Dr. Tardif’s lab and cultured in medium 231 (M-231-500) supplemented with smooth muscle growth supplement (S-007-25; Gibco). Monocytes were obtained from Dr. Rioux’s lab and we cultured them in RPMI with 10% fetal bovine serum. RNA from tissues was extracted either with the Ribopure Kit (Ambion) or using EZ1-XL Advance and Qiagen EZ1 RNA Tissue Mini Kit. hCA were obtained from the “Réseau d’Échanges de Tissus et d’Échantillons Biologiques” (RÉTEB) biorepository at the Montreal Heart Institute and Quebec Heart and Lung Institute. We purchased adult brain total RNA (540005–41, lot#6048990) and adult heart total RNA (540011–41, lot#6056165; Agilent Technologies).

Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (PromoCell, C-12200). HUVECs were cultured in an endothelial cell growth medium kit (PromoCell, C-22110) with 1% penicillin/streptomycin (Capricorn Scientific, PS-B; Ebsdorfergrund, Germany). Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta of a 7-week-old male Sprague-Dawley rat using previously published procedures [48 (link)]. Briefly, the rat was sacrificed with an overdose injection of Zoletil 50 and Rompun. Then, the rat was disinfected with povidone. The chest was opened and the thoracic aorta was dissected using micro-dissecting scissors to open the aorta and remove the endothelial layers by gentle scraping. The aorta was cut until 1–2 mm³ and treated with collagenase and elastase (Sigma, #E1250) for 2 h at 37 °C. The cells were transferred to a tube containing the cell culture medium and centrifuged. The cell pellets were washed with PBS 3 times. Finally, VSMC pellets were resuspended in the cell culture medium and cultured in a cell incubator. VSMCs were cultured in DMEM (Capricorn Scientific, DMEM-HPA; Ebsdorfergrund, Germany) with 10% FBS (Capricorn Scientific, FBS-11A; Ebsdorfergrund, Germany), 5% SMGS (Gibco, S-007-25), and 1% penicillin/streptomycin. All cells were cultured at 37 °C in a humidified environment containing 5% CO₂.