Tg mice expressing DN-RAGE in endothelium. Founders were identified by Southern blotting (A) and by PCR (B). Lanes 1 and 6, lack of PPET DN-RAGE expression; lanes 2–5, expressing PPET DN-RAGE; C, control; M, base pair size marker. (C) Aortas were retrieved from the indicated mice and subjected to Western blotting using anti-RAGE IgG. (D) Thioglycollate-elicited macrophages were retrieved from the indicated mice and were incubated with S100b for 20 minutes. Western blotting was performed to detect phospho-JNK MAP kinase (P-JNK) followed by total JNK MAP kinase (T-JNK). Where indicated by the white line, lanes were run on the same gel but were noncontiguous. (E–J) Impact of RAGE on atherosclerosis at 14 weeks. Shown are representative images of aortic arches (E–G) and sections stained with oil red O (H–J). (K and L) Hearts were retrieved from apoE^–/– (n = 12), apoE^–/–RAGE^–/– (n = 13), or apoE^–/–Tg PPET DN-RAGE (n = 7) mice, and mean atherosclerotic lesion area (K) and lesion complexity profile (L) were determined. (M) Endothelium-dependent vasorelaxation was tested in isolated mouse aortic rings from apoE^–/–, apoE^–/–RAGE^–/–, and apoE^–/–Tg PPET DN-RAGE mice (n = 5 per group) sacrificed at 14 weeks of age. Relaxation is reported as percent of initial phenylephrine precontraction. Comparisons were conducted among groups for each agonist dose. *P < 0.05, apoE^–/–RAGE^–/– versus apoE^–/– (doses >3 x 10^–7 M); **P < 0.001, apoE^–/–Tg PPET DN-RAGE versus apoE^–/– (doses >10^–8 M).