Cells were isolated from the blood vessels in rat, mouse and human. Human carotid arteries were obtained from National Disease Research Interchange (Philadelphia, PA). The cells isolation methods were described previously 56 (link). Briefly, the tissue segments were washed three times with phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S). The surrounding connective tissues and adventitia were dissected away under a dissecting microscope. Endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades. For tissue explant culture method, the tunica media was cut into mm-size and placed onto the surface coated with 1% CellStart (Invitrogen Corp.) in 6-well plates. The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R&D Systems, Inc.) (Maintenance medium). For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase (Sigma-Aldrich Inc.) in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml). After incubation at 37°C for 30 min, the same volume of 1 mg/ml elastase (Sigma-Aldrich, Inc.) solution was added to the solution containing the tissue and collagenase. The tissues were incubated for another 1–2 hours until all the tissues were digested. Cells were then seeded onto CellStart-coated dishes and maintained at 37°C in an incubator with 5% CO2.