R-VSMCs were maintained in minimum essential medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic selleck chemicals llc solution (Sigma). Cells were fed every 2 days and subcultured upon reaching 90% confluence. Human aortic VSMC (H-VSMC), human umbilical vein endothelial cells, and human aortic endothelial cells were purchased from Clonetics (Lonza, Basel, Switzerland) and maintained as recommended by the supplier. H-VSMC were cultured in smooth muscle cell basal medium supplemented with 5% FBS, 0.1% insulin, 0.2% human fibroblast growth factor, 0.1% gentamicin, and 0.1% human EGF. Growth medium was replaced every 2 days, and the cells were subcultured upon reaching 80% confluence.

Prior to each experiment, cells were seeded into multiwell plates as appropriate and incubated for 24�C48 h in serum-free growth medium supplemented with 0.1% bovine serum albumin and 1% antibiotic/antimycotic solution. All experiments on primary cells were performed between passages four and nine. HEK293 Cell Culture and Transfection HEK293 cells were obtained from the American Type Culture Collection and maintained in minimum essential medium with Earle’s salts supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution. Transient transfection of HEK293 cells with plasmids encoding GFP-tagged PAR1�C4 was performed in 6-cm dishes using FuGENE 6 (Roche Applied Science) according to the manufacturer’s instructions with 5 ��g of plasmid DNA per dish and 3 ��l FuGENE per ��g of DNA.

Prior to experimentation, transfected cells were incubated for 24 h in serum-free growth medium supplemented with 10 mm HEPES (pH 7.4), 0.1% bovine serum albumin, and 1% antibiotic/antimycotic solution. Activation of Plasma Prekallikrein Serum-deprived R-VSMC, H-VSMC, human umbilical vein endothelial cells, or human aortic endothelial cells cultured in black wall clear-bottomed 96-well plates (Corning, Inc., Corning, NY) were incubated with 100 nm human PK (Enzyme Research Laboratories, Inc., South Bend, IN) in the presence of 0.6 mm of the chromogenic substrate for plasma KK, S2302 (Chromogenix, Milano, Italy). Immediately upon addition of the reagents, plates were placed in a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA) and absorption at 405 nm was measured at 2-min intervals for 2 h.

Metalloprotease Activity ADAM activity was measured using two approaches. For the fluorogenic substrate assay, R-VSMCs at 90% confluence in 96-well plates were serum-starved for 48 h, after which the medium was replaced with Hank’s balanced salt solution containing 20 nm human plasma KK (Enzyme Research Laboratories, Entinostat Inc.) and 20 ��m of the TACE II fluorogenic peptide substrate, MCA-KPLGL-Dpa-AR-NH2 (EMD Biosciences; Gibbstown, NJ).