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. 2005 Aug 10;102(34):12165–12170. doi: 10.1073/pnas.0500641102

Fig. 1.

Fig. 1.

Angiogenesis in aortic rings in vitro. (A) Light microscopy of new vessels shown sprouting from aortic rings. (i) Aortic ring incubated for 7 days without glucocorticoid. (ii) Aortic ring incubated for 7 days in the presence of glucocorticoid. Thick white arrows indicate the aortic ring; thin white arrows indicate new vessels. (Scale bar: 0.2 mm.) (iii) Uptake of low-density lipoprotein (LDL) is shown by fluorescence microscopy. This ring was incubated for 7 days without steroids. Thick white arrows indicate the aortic ring; thin white arrows indicate uptake of fluorescent labeled LDL in endothelial cells in new vessels; black arrows indicate uptake in endothelial cells of the aortic ring. (Scale bar: 0.2 mm.) (iv) High power view of new vessels; thick white arrows indicate the aortic ring and thin white arrows indicate uptake of fluorescent labeled low density lipoprotein in endothelial cells (Scale bar: 0.02 mm.) (Bi) Time course and effect of corticosterone on angiogenesis. ○, results from vessels incubated without steroids; ♦, results from vessels incubated with corticosterone (600 nM). Results are mean ± SEM for n = 4 per group. Comparison was by repeated measures ANOVA; *, P < 0.02. (Bii) Effects of corticosterone and 11-dehydrocorticosterone. Vessels were counted after 7-day incubation with steroids. Results are mean ± SEM. #, P < 0.01 versus vehicle by 2-way ANOVA and least squares difference post hoc test. (C) Influence of receptor antagonism. (i) Effects of the mineralocorticoid receptor antagonist spironolactone. Aortic rings from C57Bl6J mice were incubated with (filled bars) and without (open bars) spironolactone (10-6 M) and glucocorticoids (600 nM). Results are mean ± SEM for n = 6 experiments. #, P < 0.02 versus corresponding vehicle. Spironolactone alone had no effect. (ii) Effects of the glucocorticoid receptor antagonist RU38486. Aortic rings from C57Bl6 mice were incubated with (filled bars) and without (open bars) RU38486 (10-6 M) and glucocorticoids (600 nM). Results are mean ± SEM for n = 4-6 experiments. # P < 0.01 versus corresponding vehicle. ***, P < 0.001 for the effect of RU38486 in the presence of glucocorticoid. RU38486 alone had no effect. (D) Effects of 11βHSD inhibition (i) Pharmacological inhibitor carbenoxolone. Aortic rings from C57Bl6J mice were incubated with (filled bars) and without (open bars) carbenoxolone (10-6 M) and glucocorticoids (600 nM). Results are mean ± SEM for n = 5 experiments. #, P < 0.01 versus corresponding vehicle. *, P < 0.04 for the effect of carbenoxolone in the presence of 11-dehydrocorticosterone. Carbenoxolone had no effect in the presence of corticosterone or vehicle alone. (ii) Transgenic deletion of 11βHSD1. Effects of corticosterone and 11-dehydrocorticosterone on angiogenesis in vessels from 11βHSD1 -/- mice. Aortic rings from C57Bl6J wild-type (open bars) or 11βHSD1 -/- (filled bars) mice were incubated with and without glucocorticoids (600 nM). Results are mean ± SEM for n = 7 experiments. #, P < 0.01 versus corresponding vehicle. **, P < 0.01 for differences in angiogenesis between wild-type and 11βHSD1 -/- mice. Angiogenesis was not different between strains in the presence of vehicle or corticosterone but was inhibited by 11-dehydrocorticosterone in wild-type but not 11βHSD1 -/- mice.