Pcs 100 021

A7r5 rat aortic SMCs were obtained from American Type Culture Collection (ATCC). A7r5 cells were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). Cells were maintained in 37°C and 5% CO₂ environment. Only cells from passages 3–8 were used in experiments. For LPA treatments, A7r5 cells were quiesced in low-glucose (1 g/L) and low-FBS (0.1% FBS) media for 24 hours prior to LPA treatment. The treatment was then removed, and the cells were either immediately stimulated with 30 μM LPA for 30 minutes or cultured for an additional 1, 3, 7, or 10 days in low-glucose, low-serum media prior to LPA stimulation. Primary rat aortic SMCs were collected from 4 male Wistar rats (The Jackson Laboratory), endothelium was removed by swabbing, and the remaining tunica media was dissociated (Miltenyi Biotec tissue dissociation kit) according to the manufacturer’s instructions for 40 minutes. Subsequently, cell suspensions were forced through a 70 μm strainer washed, pooled, and cultured in a T-75 flask in DMEM with 20% FBS and 1% P/S until 80%–90% confluent. Primary human coronary artery SMCs from 3 human donors were obtained from ATCC (PCS-100-021). Human coronary artery SMCs were cultured using vascular cell basal medium (ATCC, PCS-100-030) supplemented with a VSMC growth kit (ATCC, PCS-100-042) and 20% serum to induce phenotype switching.