In situ superoxide detection with MCLA in aortas. Shown are representative photographs for chemiluminescent signal of MCLA (d–f) and each corresponding aorta (a–c) that was stained with Sudan III from WT mice (a and d), apoE-KO mice (b and e), and apoE-KO/eNOS-Tg mice (c and f). The chemiluminescent signal due to superoxide production was more widely distributed and more strongly visualized in apoE-KO/eNOS-Tg mice compared with apoE-KO mice. (g) Quantitative analysis for superoxide levels in nonplaque and plaque areas in WT mice (white bar), apoE-KO mice (black bars), and apoE-KO/eNOS-Tg mice (gray bars). Superoxide levels were expressed as the ratio (fold increase) to the control values obtained from the normal aortic vessels of WT mice. Superoxide production in apoE-KO/eNOS-Tg mice was significantly increased in both nonplaque and plaque areas compared with apoE-KO mice. n = 6–10 for each group. *P < 0.05 vs. normal vessels in WT mice; **P < 0.05 vs. nonplaque areas in apoE-KO mice; ***P < 0.01 vs. plaque areas in apoE-KO mice. (h) Reduction of superoxide levels by endothelial denudation in nonplaque and plaque areas of apoE-KO mice (black bars) and apoE-KO/eNOS-Tg mice (gray bars). Shown is the percentage of decreased superoxide production from each predenuded level. In apoE-KO/eNOS-Tg mice, removal of the endothelium resulted in significantly marked decreases in superoxide production in nonplaque areas of the vessels. n = 5–6 for each group. *P < 0.01 vs. nonplaque areas in apoE-KO mice.