Alterations in endothelial cell proliferation and apoptosis contribute to vascular remodeling following hind-limb ischemia in rabbits

Hind-limb ischemia is a potent stimulus for angiogenesis. However, capillary density does not change in tibialis anterior muscle (TA) following hind-limb ischemia, despite increases in angiogenic growth factors. The objective of this study was to determine whether changes in proliferation and apoptosis occurred in the same muscle. In total, 19 New Zealand white rabbits underwent femoral artery ligation and excision and the ischemic and contra-lateral (control) TA muscles were harvested after 1 (n = 7), 5 (n = 7) and 21 (n = 5). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to detect apoptosis and double staining was used to identify the apoptotic cell types. Proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen (PCNA) and [H-3]thymidine incorporation, in vitro. TUNEL positive nuclei were greater in ischemic than control muscle at 1 day (1.83 +/- 0.70% vs 1.03 +/- 0.20%), 5 days (2.13 +/- 0.50% vs 1.21 +/- 0.42%) and at 21 days the difference was statistically significant (3.42 +/- 0.80% vs 0.96 +/- 0.40%, p < 0.01). The majority of TUNEL positive nuclei were endothelial (Tie2 positive) cells. The number of PCNA positive cells in ischernic versus control muscle was similar at 1 day (0.71 +/- 0.20% vs 0.53 +/- 0.20%) and 5 days (1.28 +/- 0.30% vs 0.77 +/- 0.30%), but was significantly (p < 0.05) reduced in ischernic muscle at 21 days (0.18 +/- 0.20% vs 1.35 +/- 0.30%) with no difference in [H-3]thymidine incorporation. Directionally opposite changes in endothelial cell proliferation and apoptosis occur in TA muscle following hind-limb ischemia. Modulating apoptosis in ischemic skeletal muscle may present a novel therapeutic target in peripheral arterial disease.