Figure 5. Hcy derived ROS trigger endothelial casp1 activation.

HUVECs were cultured in 6cm dishes as in Fig.1. A&B, Cells were transduced by adenoviral ec-SOD (1 MOI) for 48hr, treated with PEG-catalase (25mg/mL) for 30m with refreshed medium, and then with L-Hcy (500µM) and/or LPS (10 µg/mL) for additional 24hr before subjected for cell death and casp1 activity analysis. A, Pyroptosis/apoptosis (anti-oxidants rescue, FCM). B, Casp1 activity (anti-oxidants rescue, FCM). C, H₂O₂ on Casp1 activity (casp1 inhibition rescue, FCM). Cells were pretreated with casp1 inhibitor or inhibitor of cathepsin B (a lysosomal cysteine protease) for 30m, and then treated with H₂O₂ (500 µM) for additional 24 hours before subjected for casp1 activity analysis. Numbers above on each bar is the percentage normalized by the mean of control. Data are representative of 4 separated experiments and presented as Mean±SEM. #, P<0.05 vs vehicle in A, B; *, P<0.05 vs non-treatment control; †, P<0.05 vs H₂O₂ treatment alone in C. Arrows indicate the direction of significant changes. Values are Mean±SEM; n=4.