Loss of endothelial basal NO activity, prior to the presence of media calcifications. Aortic contractility, assessed by the addition of phenylephrine (PE, 3 nM–10 μM) to the organ bath. Two weeks: the concentration–response curve for PE is shown in the absence of N(ω)-nitro-L-arginine methyl ester (–LN) (A) and in the presence of LN (+LN) (B). Additionally, the LN effect was calculated (C). Four weeks: identical to previous timepoint (D–F). Number of animals per group (A–F): –LN, 2 weeks control: n = 6; –LN, 4 weeks control: n = 5; +LN, 2 and 4 weeks control/adenine: n = 6; ΔLN, 2 weeks control/adenine: n = 6; ΔLN, 4 weeks control/adenine: n = 5. Two-way ANOVA with Bonferroni multiple comparison test (A–F): P > .05: not significant, not shown; **P < .01; ***P < .001; ****P < .0001. Data represented as mean ± SEM.