eNOS plays a critical role in RANKL-induced angiogenesis. (A) Aortic segments were harvested from WT and eNOS KO mice (n = 7 per group). Endothelial-cell sprouts forming branching cords from the margins of vessel segments taken from mice were photographed under a phase microscope. Staining of endothelial cells sprouted from RANKL-treated aorta with VWF (middle). (B) Sprouting scores were classified from 0 (least positive) to 5 (most positive). Data are means ± SEs. (C-E) WT and eNOS KO mice (n = 7 per group) were injected with 0.6 mL Matrigel containing RANKL (10 μg). After 7 days, the mice were killed and the Matrigel plugs were excised. (C) Plugs were stained for infiltrating endothelial cells using anti-CD31 antibody. Arrows indicate CD31⁺ cells. (D) Quantitative assessment of CD31⁺ endothelial cells. (E) Quantification of neovessel formation by measuring hemoglobin in the Matrigel. Data are means ± SDs; *P < .05; * *P < .01 versus RANKL in WT.