Standard laboratory chow

The animal care and use procedures were approved by the Animal Care Committee of the University of Tokyo (Permit Number: P09-015). Male C57 BL mice were purchased from Oriental Yeast (Tokyo, Japan) and fed standard laboratory chow (CLEA Japan, Tokyo, Japan). After drug treatment for 7 days, the mice were sacrificed to extract the descending aortas. The tissues were fixed in 4% paraformaldehyde, dehydrated, and then embedded in paraffin. The paraffin-embedded tissues were cut into cross sections (2 µm). The sections were deparaffinized and rehydrated for immunohistochemistry. Tissue sections were incubated with rabbit anti-phospho eNOS (Ser1177) or -eNOS antibodies diluted at 1:200, followed by a biotinylated anti-rabbit secondary antibody and then horseradish peroxidase-labeled streptavidin, according to the manufacturer's instructions (DAKO, Copenhagen, Denmark). Staining intensities were measured in 30 randomly selected fields of the endothelium and media by Scion Image program. The intensity ratio of the endothelium to media was then compared statistically.

Male ICR mice (5 weeks old) weighing 25–30 g (Charles River Laboratories Japan, Yokohama, Japan) maintained at 25 °C and 55% humidity were allowed free access to standard laboratory chow (Clea Japan, Tokyo) and water. They were fasted for 24 h prior to and during the experiment but were allowed free access to water. Our study was carried out in accordance with the Guidelines for Laboratory Animals of Okayama University (OKU-2016150).