Figure 2.

PDI upregulation promotes simultaneous increases in proliferation and apoptosis in the diabetic vein graft atherosclerosis. Paraffin-embedded sections of the vein grafts of non-diabetic and diabetic mice killed 8 weeks after surgery were used for: (a–f) triple-labeling immunofluorescence with primary Ki67 antibody and TUNEL kit and Cy3-conjugated secondary antibody was performed and counterstained with DAPI; (a, d) DAPI staining shows cell nuclei (blue) in the walls of vein grafts from non-diabetic and diabetic mice. An akaryotic area can be seen through the medium of the diabetic vein grafts; (b, e) merged images from Ki67-positive (red) and TUNEL-labeling (green) cells in the walls of vein grafts from non-diabetic and diabetic mice; (c, f) Merged images show co-distribution of proliferating cells (red, Ki67-positive) and apoptotic cells (green, TUNEL-labeling) in the walls of vein grafts from (a, b, d and e); (g, h) statistical results show ratios of Ki67- and TUNEL-positive cell numbers/ total cell numbers from (a to f) and from three independent experiments. a, P<0.05 versus non-diabetic mice as indicated by analysis of variance (ANOVA) with LSD test. Data are shown as means±SEM. (i–x) Triple-labeling immunofluorescence with primary PDI and Ki67 antibodies or TUNEL kit and Cy3-conjugated secondary antibody was performed and counterstained with DAPI. (i, m, q and u) DAPI staining shows cell nuclei (blue) in the walls of vein grafts from non-diabetic and diabetic mice. (j, n, and r, v) PDI staining (j, n, green; and r, v, red) in the cells of vein grafts from non-diabetic and diabetic mice; (k, o) Ki67-positive cells (red, proliferating cells) and (s, w) TUNEL-positive cells (green, apoptotic cells) in the vein grafts from non-diabetic and diabetic mice; (l, p) Merged images show co-distribution of PDI-labeling cells (green) and proliferating cells (red, Ki67-positive) and (t, x) PDI-labeling cells (red) and apoptotic cells (green, TUNEL-labeling) in the walls of vein grafts from non-diabetic and diabetic mice. Scale bar, 100 μm. Arrowheads and stars indicate the VSMCs thickness and lumens of the vein grafts, respectively