Fig. 4.

Caspase-3 promotes endothelial barrier integrity during thrombin-induced disruption. Human lung microvascular endothelial cells (HLMVECs) were exposed to 1.25 U/ml of thrombin, and endothelial barrier integrity was measured. A and B: HLMVECs grown in basal media or serum-free media (2.5 and 0% fetal bovine serum, respectively) show a drop in transendothelial resistance (TER) in response to thrombin. Caspase-3 inhibition with z-DEVD-FMK (DEVD) leads to a more significant drop in TER compared with thrombin alone. Summation of all individual wells for each condition is plotted. C: quantification of maximal drop in TER. D: HLMVECs were grown in 6-well plates and treated with nontargeting small-interfering RNA (siRNA) or siRNA against caspase-3. Following siRNA exposure (24 h), HLMVECs were plated on Electrical Cell-substrate Impedance Sensing System (ECIS) plates; an aliquot of cells was replated for immunoblotting. Cells were harvested at 48 h after siRNA treatment, at the time of ECIS experiments. Left, immunoblotting for caspase-7 shows no nonspecific knockdown of caspase-7 expression. Right, immunoblotting for caspase-3 shows significant knockdown of caspase-3 expression. E: quantification of maximal drop in TER shows knockdown of caspase-3 leads to significant thrombin-induced endothelial barrier disruption; n = 3–4 separate experiments; 6–14 individual wells/condition.