Arterial smooth muscle cells (ASMCs) were obtained from medium of internal mammary arteries, widely used for vascular revascularization. ASMC cultures are characterized by a hill-and valley morphology.
Two-dimensional gel electrophoresis (2DE) analysis was utilized to characterize the proteome and secretome of human arterial smooth muscle cells.
The largest class of proteins identified by 2DE falls into the group of cytoskeletal components, which regulate actin polymerization allowing SMCs to control their shape and their contractile properties.
ASMCs were isolated from a residual segment of human internal mammary arteries obtained from patients undergoing coronary artery bypass grafting. The medium was stripped from the underlying adventitia, minced and digested in 5mL of HAMF10 containing collagenase type I (3 mg: 235 U/mg), elastase (7 mg: 3.73 U/mg) and soybean trypsin inhibitor (5mg) for 45min at 370C. The enzymatic activity was quenched by adding fetal calf serum (30%). Cultures of ASMCs collected from 11 individual patients were grown separately in HAM-F10 medium supplemented with FCS (30%), HS (10%), penicillin (100 units/mL), streptomycin (100 mg/mL), fungizone (2.5 U/mL), HEPES buffer (20mmol/L), insulin (2 mg/mL), and nonessential amino acid solution (1%). Medium was changed every 3 days. Cells were trypsinized at confluence and reseeded at a 1:2 ratio. Confluent cells at passage 2 were washed three times with Hank’s Balanced Salt Solution buffer, and then incubated for 24 h in serum-free culture medium before proteins were extracted.