Warfarin treatment of apoE^−/− mice increases apoptosis in atherosclerotic plaque.

<p>Vascular calcification is closely linked to apoptosis. Therefore we stained sections for caspase-3. Moreover, all animals were injected with annexin A5-biotin, a protein with high affinity for phosphatidylserine (PS). Both cleaved caspase-3 (A,C) and annexin A5-biotin (B,D) increased in the warfarin treatment, with annexin A5 accumulation significantly increased after 4 weeks of warfarin treatment (B). Annexin A5 staining using AP vector blue confirmed PS externalization at different sites in the atherosclerotic plaques. Activated and apoptotic macrophages are known to express PS on their surface. We found co-localization of macrophages in the shoulder of the atherosclerotic plaque (Mac3; F) and PS (annexin A5 stained with AP vector red; E), indicating activated and apoptotic macrophages. In the inset annexin A5 (blue) localizes with Mac-3 staining (red/brown), indicating activated macrophages. Moreover, at baseline annexin A5 was only incidentally present at the surface of the plaque (G), whereas at the 4 weeks warfarin treatment, annexin A5 stained significantly positive (H). Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05. i, intima; m, media; l, lumen.