Ang 2

High-glucose Dulbecco's Modified Eagle Medium (DMEM) with l-glutamine, phenol red, sodium pyruvate, and penicillin-streptomycin solution was purchased from Wako (Japan). Fetal bovine serum (FBS) of South American Origin was purchased from Corning (NY, USA). Herpud1 Silencer Pre-designed siRNA was purchased from Ambion Thermo Fisher Scientific (Waltham, MA, USA). Herpud1 plasmid was purchased from OriGene (MD, USA). Ang II was purchased from Wako (Japan).

Apoe^−/− mice (C57BL/6J background) and Tlr9^−/− mice (C57BL/6J background) were originally purchased from the Jackson Laboratory and Oriental BioService, Inc, respectively. Tlr9^−/−Apoe^−/− mice were generated by crossing Apoe^−/− mice and Tlr9^−/− mice. Male mice were fed a Western‐type diet (WTD; Oriental Yeast Co Ltd) from 6 weeks of age through the completion of the experiment. Ang II, 1000 ng/kg per minute (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was infused by an osmotic pump (Alzet, Cupertino, CA) from 2 weeks after starting a WTD, for 4 weeks. For in vivo TLR9 inhibition, phosphothioate‐modified oligodeoxynucleotide—iODN2088 (5′‐tcctggcggggaagt‐3′) was used. Control (Ctrl)‐iODN2088 (5′‐tcctgagcttgaagt‐3′) was used as its control. These oligodeoxynucleotides were synthesized with a low level of endotoxin (<0.5 endotoxin units/mg; Gene Design Inc., Osaka, Japan) and intraperitoneally injected into Ang II–infused Apoe^−/− mice (150 μg) three times a week for 4 weeks. All mice were housed under a 12‐hour light/dark cycle, with food and water available ad libitum. All experimental procedures conformed to the guidelines for animal experimentation of Tokushima University. The protocol was reviewed and approved by our institutional ethics committee.