Ptdcho

HASMCs (cat. no. KS-4009; Kurabo Biomedical, Osaka, Japan) were maintained in Dulbecco s modified Eagle s medium (DMEM; high glucose; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) containing smooth muscle growth supplement (SMGS; Cascade Biologics, Tokyo, Japan) , 10％ FBS (Biological Industries, Kibbutz Beit-Haemek, Israel) , and antibiotics (100 U/mL penicillin, 100 μg/ mL streptomycin, and 0.25 μg/mL amphotericin B, all purchased from Gibco, Invitrogen Co., Grand Island, NY, USA) . HASMCs at passages 6-7 were cultured in a humidified atmosphere containing 5％ CO 2 in air at 37℃. PtdCho and lysoPtdCho (purity ≥ 99％ ) ( Fig. 1A) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ethanol to prepare stock solutions at a concentration of 40 mM) . To examine the effect of lysoPtdCho on the proliferation of HASMCs, cells were cultured in DMEM containing SMGS for 24 h in a 6-well plate at 5×10 5 cells/well. After 24 h, the medium was replaced with DMEM without SMGS, and the cells were further incubated for 24 h. Then, the cells were treated with PtdCho or lysoPtdCho (1, 10, 25, or 50 μM) in DMEM containing 1％ or 10％ FBS for 24 h. Control groups were treated with phosphate-buffered saline (PBS) . Cell viability was determined using Cell Counting Kit-8 (CCK-8; DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer s instructions.

PtdSer derived from bovine brain or soybean (Glycine max; purity ≥ 98％) and phosphatidylcholine (PtdCho) (purity ≥ 98％; both from Sigma-Aldrich, St. Louis, MO, USA) were dissolved in chloroform/methanol (90:10, v/v) . PSL was prepared from a lipid mixture of PtdSer (14 mM) and PtdCho (33 mM) at a molar ratio of 3:7, with or without the fluorescent dye, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) ( ammonium salt; Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) . The solvent was removed under nitrogen with stirring and dried in a desiccator overnight. The liposomal pellet was then resuspended in PBS (10 mg/mL) .