Smooth muscle growth medium 2

HASMCs were cultured in smooth muscle growth medium-2 (Lonza), which contains fetal bovine serum (FBS) and various growth factors, and starved for 24 h before stimulation in smooth muscle basal medium-2 (Lonza) devoid of serum or growth factors. Raw264.7 cells were purchased from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. BMDMs were isolated from femurs of wild-type mice and cultured in Mφ medium (RPMI 1640 medium supplemented with 10% FBS, 40 ng ml⁻¹ murine Macrophage Colony-Stimulating Factor (M-CSF), 2 mM L-glutamine, 50 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin). For M1 or M2 polarization, Raw264.7 cells or BMDMs were treated with LPS (50 ng ml⁻¹) or IL-4 (20 ng ml⁻¹) for 24 h, respectively. CM of Raw264.7 cells or BMDMs were collected after another 24 h.

hiPSC-derived VSMCs were purchased from ImStem Biotechology (Farmington, CT, USA). hiPSC-derived VSMCs were chosen to ensure an adequate amount of the same cells for all experiments. Primary rat VSMCs were prepared by using the enzymatic digestion of thoracic arteries from 3-week-old Sprague–Dawley rats. Transfection reagents were obtained from ThermoFisher Scientific (Waltham, MA, USA), except for siRNA targeting rat ENPP1, which was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cells were seeded in a collagen type I-coated 60 mm dish or a regular polystyrene dish at a density of 3500 cells/0.32 cm² in Smooth Muscle Growth Medium-2 (Lonza, Allendale, NJ, USA) or Vascular Cell Basal Medium (ATCC, Manassas, VA, USA), which is contained in the Vascular Smooth Muscle Cell Growth Kit (ATCC). After overnight culture, either siRNA targeting human ENPP1 s10264 (Cat 4390824) and control siRNA (Cat 4390846) or siRNA targeting rat ENPP1 (SASI Rn01_00111206) and control siRNA (Cat 4390847) were transfected into hiPSC-derived VSMCs or rat VSMCs, respectively, using Lipofectamine RNAiMAX overnight, according to the manufacturer’s instructions.

THP-1 cells, an immortalized human monocyte cell line, were grown in suspension in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin (Pen-Strep, Gibco) at a density of 1 × 10⁶/ml. For macrophage differentiation, THP-1 cells were plated at 2 × 10⁵ cells/ml in PMA-containing medium (phorbol 12-myristate 13-acetate at 1 × 10⁻⁷ M) for 72 h and then switched to PMA-free medium, rested for 24–48 h prior to use. THP-1 macrophages were found to be HNR for the rs3045215 variant after genotyping with the BsrI/PCR method (12 (link)) with DNA isolated by the HotSHOT method (32 (link)).
Adult primary HAoSMC (Cell Applications) from healthy human donors were grown in SmGm-2 medium (Lonza, Smooth Muscle Growth Medium-2) with 5% FBS and SingleQuots supplements (Lonza, human epidermal growth factor, human fibroblastic growth factor, insulin, and gentamicin/amphotericin-B). Cells were split at 80% confluence using Trypsin-EDTA (Gibco, 0.05%, containing phenol red) and medium was renewed every 2 days. DNA was extracted using the HotSHOT method (32 (link)). HAoSMC rs3045215 genotypes were as follows: homozygous non-risk (non-deletion), HNR = 1,441, 1,473 and 3,003; heterozygous, HET = 1,596 and 2,228. No homozygous risk (deleted) HR HAoSMC was identified by genotyping.