Up-regulation of ADAM10/17 activity by proinflammatory mediators. (A) HUVECs were stimulated with IFN-γ and TNF-α (each 10 ng/mL) or vehicle control (PBS) for 16 hours and analyzed for ADAM10 and ADAM17 surface expression by flow cytometry. Data are shown as representative histogram (left) or as mean plus or minus SD of the fluorescence signals determined for ADAM10 and ADAM17 determined in 3 independent experiments (right). Surface expression was expressed in relation to that determined for the PBS-treated control. (B) HUVECs were stimulated with IFN-γ and TNF-α (each 10 ng/mL) or vehicle control (PBS) for 16 hours, and analyzed for cell-associated ADAM10/17 activity using a fluorogenic peptide-based assay. The shedding activity (mean plus or minus SD) was determined in 3 independent experiments using a recombinant ADAM17 standard run in parallel and expressed in relation to that of the vehicle-treated control. (C,D) Isolated murine aortas were treated with vehicle control (PBS, n = 4) or IFN-γ and TNF-α (each 20 ng/mL) in the absence (n = 4) or presence (n = 3) of the combined ADAM10/17 inhibitor GW280264 (GW, 5 μM) for 16 hours. Subsequently, cell lysates were analyzed for ADAM10/17 activity (C), and conditioned media were assayed for release of JAM-A into the culture medium (D). *Significant increase compared with the vehicle-treated controls (P < .05).