Figure 2.

EVA1A (eva-1 homolog A) promotes endothelial apoptosis under proatherogenic disturbed flow. A through C, Human umbilical vein endothelial cells (HUVECs) were treated with EVA1A siRNA (small interfering RNA) or with nontargeting control (NTC) siRNA before exposing to flow for 72 h using the orbital shaker system. A, ECs were isolated from disturbed flow (DF) and undisturbed flow (UF) regions and the efficiency of EVA1A siRNA was assessed by quantitative real-time polymerase chain reaction (qRT-PCR; n=4 donors) using HPRT as a housekeeping gene. EVA1A relative expression was normalized to UF NTC siRNA. B and C, EC apoptosis under DF and UF was assessed by immunostaining using antiactive caspase 3 antibody (C, green) and costaining with EC marker CDH5 (cadherin 5; C, red) and DAPI (4′,6-diamidino-2-phenylindole; C, blue). Apoptotic ECs are indicated with white arrows. The graph in B represents percentage of apoptotic ECs calculated by dividing the number of active caspase 3-positive cells by the total number of EC per field of view (n=4 donors). D and E, HUVECs were treated with EVA1A siRNA or with nontargeting control (NTC) siRNA and cultured on transwell inserts. ECs were exposed to DF for 72 h using the orbital shaker system before assessment of endothelial permeability under static conditions for 1 h using rhodamine (Rd)-albumin as a tracer. The graph in E represents concentration of Rd-albumin measured in the lower compartment (n=6). A, B, and E, Data are presented as means±SEM. Differences between groups were analyzed using a 2-way ANOVA (A and B) with Tukey post hoc test or unpaired t test (E) and P values are shown in the graphs. Scale bar: (C), 100 µm.