Figure 6.

Endothelial nitric oxide (NO) synthase expression and phosphorylation in aorta. A and B: Western blotting was used to measure the expression and phosphorylation of endothelial nitric oxide synthase (eNOS) and phosphorylation of AKT (p-AKT) in the aorta from mice exposed to 16 and 60 wk of either air or electronic cigarette (e-cig) vape generated from liquid containing nicotine (NIC) 0 mg/mL (ECV-0), 6 mg/mL (ECV-6), or 24 mg/mL (ECV-24). C and D: quantitation of eNOS bands in A and B, respectively, showing downregulation of e-NOS in an exposure time- and NIC-dependent manner. E and F: quantitation of p-eNOS bands in A and B, respectively, showing similar decreases as in C and D. G and H: quantitation of p-AKT bands in A and B, respectively, also exhibiting similar decreases. I: frozen aortic sections were incubated with primary antibody against eNOS and p-eNOS followed by corresponding secondary fluorescence antibody (green) and the nuclear stain DAPI (blue). J: quantitation of green fluorescence of eNOS and p-eNOS. ECV exposure downregulated eNOS expression and decreased Akt-dependent eNOS phosphorylation, contributing to the decline of NO synthesis and onset of VED. Data are presented as means ± SE of 6 experiments. For C–H, data are presented as means ± SE of independent experiments in 6 mice, whereas for J, each point is based on independent experiments in 4 mice. Analysis was done using two-way ANOVA followed by Bonferroni multiple-comparisons test. The differences were considered statistically significant at P ≤ 0.05. *Significant from air-exposed controls at P < 0.05; #significant from ECV-0 at P < 0.05; @significant from ECV-6 at P < 0.05; $significant from the same exposure at 16 wk at P < 0.05. †significant different from eNOS expression at P < 0.05.