Effect of different dietary treatments on apoptotic activity at the 12-week time point. Sections were prepared as described in the legend of Figure 2. Longitudinal sections of each thoracic aorta were stained by immunohistochemical techniques with an antimacrophage antibody and with a terminal dUTP nick-end labeling (TUNEL) staining for apoptosis (see “Materials and methods” for details). Panel A represents 12 weeks of normal vitamin K diet (5 μg/g K₁), panels B and E represent 12 weeks of W&K diet (3 mg/g warfarin and 1.5 mg/g vitamin K₁), panel C represents normal K (5 μg/g) after 6 weeks of W&K, and panels D and F represent 6 weeks of high vitamin K (100 μg/g K₁ or K₂) after 6 weeks of W&K. Macrophages are absent in the medial layer of aortic tissue, both in rats treated with W&K (E) and high vitamin K (F). Also, in control animals macrophages were absent (data not shown). In panel E a positive control is shown as an inset (arrow indicates macrophage). The W&K-treated animals (B), however, have significantly increased apoptosis of vascular smooth muscle cells (VSMCs) compared with control animals (A), in which apoptosis is hardly measurable. Also in the normal K-treated animals, after 6 weeks of W&K, (C) apoptosis is clearly visible. The rats treated with high vitamin K (D) showed significantly reduced apoptosis as compared with the normal-treated animals (C). See “Results” for details. Magnification, × 400. Details of image acquisition are provided in the legend of Figure 2.