Impairment of RANKL-induced vascular hyperpermeability in eNOS-deficient mice. (A) HUVECs were preincubated for 30 minutes with or without NMA (1 mM) and stimulated with 5 μg/mL RANKL for 1 hour. A [³H]sucrose permeability assay was then performed. Three independent experiments were performed in duplicate. Data are means ± SEs; * *P < .01 versus RANKL alone. (B) An in vivo Miles vascular permeability assay was performed in WT and eNOS KO mice (n = 7 per group) as described in Figure 1. Data are means ± SDs; * *P < .01 versus RANKL in eNOS KO. (C) Representative fluorescence images of retinal vessels. RANKL (10 μg) or PBS was injected into the vitreous cavity of WT and eNOS KO mice (n = 7 per group). After 24 hours, the mice received an intravenous injection of 10 mg FITC-dextran (MW = 20 000 D), and their retinas were flat-mounted. (D) The vascular permeability was quantified by counting sites with extravasation of fluorescence at postcapillary vessel. Data are means ± SDs; * *P < .01 versus RANKL in eNOS KO. (E-F) HUVECs were preincubated for 30 minutes with or without NMA (1 mM) and stimulated with 5 μg/mL RANKL for 1 hour. (E) An immunofluorescence analysis of VE-cadherin. Arrows indicate disruption of VE-cadherin. (F) Translocation of VE-cadherin was assessed as described in “Materials and methods.” The Triton X-100–insoluble and soluble fractions were subjected to SDS–polyacrylamide gel electrophoresis followed by Western blot analysis with anti–VE-cadherin. Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of VE-cadherin to actin. VE indicates VE-cadherin; A, actin; M, membrane; C, cytosol. Data are means ± SDs; *P < .05 versus RANKL alone; #P < .05 versus untreated control.