Fig. 4.
The NADPH oxidase-ROS pathway plays a crucial role in the harmful effect of IS on valsartan-mediated NO production and tube formation in HAECs. (A–C) HAECs were treated with IS (240 μg/mL) for the indicated times. The membrane-permeable probe hydroethidine and 2′,7′-dichlorofluorescin diacetate were used to examine intracellular ROS levels. (D) HAECs were pretreated with or without the NAD(P)H oxidase inhibitor apocynin (150 μM) for 2 h and then with IS (240 μg/mL) for 60 min. Intracellular ROS levels and NADPH oxidase activities were analyzed by the intensities of the red fluorescent ethidium (ETH) product and the green fluorescent product dichlorofluorescin (DCF) and the NADP+/NADPH assay kit, respectively. (E) Cells were preincubated with apocynin (150 μM) for 2 h and then with IS (240 μg/mL) for 9 h or 18 h. Cellular lysates were subjected to Western blot analysis to evaluate the phosphorylation of PKCα and eNOSThr495. Cells were incubated with the indicated treatment agent and (F) the levels of nitrite in the culture medium and (G) tube formation was assessed. Data are expressed as the mean ± SEM. *P < 0.05 vs. vehicle-treated cells. #P < 0.05 vs. IS-treated cells, &P < 0.05 vs. IS + valsartan-treated cells.