Preventing local regeneration of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1 enhances angiogenesis

Abstract

Angiogenesis restores blood flow to healing tissues, a process that is inhibited by high doses of glucocorticoids. However, the role of endogenous glucocorticoids and the potential for antiglucocorticoid therapy to enhance angiogenesis is unknown. Using in vitro and in vivo models of angiogenesis in mice, we examined effects of (i) endogenous glucocorticoids, (ii) blocking endogenous glucocorticoid action with the glucocorticoid receptor antagonist RU38486, and (iii) abolishing local regeneration of glucocorticoids by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). Glucocorticoids, administered at physiological concentrations, inhibited angiogenesis in an in vitro aortic ring model and in vivo in polyurethane sponges implanted s.c. RU38486-enhanced angiogenesis in s.c. sponges, in healing surgical wounds, and in the myocardium of mice 7 days after myocardial infarction induced by coronary artery ligation. 11betaHSD1 knockout mice showed enhanced angiogenesis in vitro and in vivo within sponges, wounds, and infarcted myocardium. Endogenous glucocorticoids, including those generated locally by 11betaHSD1, exert tonic inhibition of angiogenesis. Inhibition of 11betaHSD1 in liver and adipose has been advocated to reduce cardiovascular risk in the metabolic syndrome: these data suggest that 11betaHSD1 inhibition offers a previously uncharacterized therapeutic approach to improve healing of ischemic or injured tissue.

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.

Fig. 2.

Angiogenesis in s.c. implanted sponges. (A) Light microscopy of hematoxylin/eosin stained sponge 8-μm sections from wild-type mice: vehicle (i) and cortisol-treated (ii) sponge (Scale bar: 400 μm) and vehicle-treated sponge at high power (iii) (Scale bar: 50 μm.) Sponges were covered with a fibroblast-rich fibrous coat and were infiltrated with inflammatory neutrophils and lymphocytes. Placebo-treated sponges alone were also infiltrated with an organized matrix and an abundance of blood vessels (black arrows). S denotes sponge matrix. (Bi) Sponges from C57Bl6J wild-type (n = 6) mice. Exposure to RU38486 or adrenalectomy (filled bars) compared with placebo or sham surgery (open bars). Results are mean ± SEM. #, P < 0.01 versus vehicle; ^*, P < 0.02 versus sham. New vessel formation was greater in RU38486-impregnated sponges or sponges from adrenalectomized mice versus their relevant controls. (Bii) Sponges from C57Bl6J wild-type (open bars, n = 12) or 11βHSD1 -/- (filled bars, n = 6) mice with and without glucocorticoids. Results are mean ± SEM. #, P < 0.001 versus corresponding vehicle. ^***, P < 0.001 for differences between wild type and 11βHSD1 -/-. Placebo-impregnated sponges exhibited an increased angiogenic response in 11βHSD1 -/- compared to wild-type mice. Cortisol inhibited angiogenesis in both strains, but cortisone inhibited angiogenesis only in wild-type mice.

Fig. 3.

Effect of injury on angiogenesis in mouse myocardium and skin. (A) Light microscopy (×50) of anti-von Willebrand factor immunostaining with fast red chromogen substrate in day 7 wild-type sham (i) and infarcted (ii) hearts. Scattered medium and large vessels were detected in sham hearts. In contrast, many more vessels were observed in the healing myocardium after infarction. Black arrows, vessels; lv, left ventricle, es, endocardial surface. (Scale bar: 100 μm.) (iii) Medium power (×100) light microscopy of hematoxylin/eosin staining in day-7 wild-type infarcted heart. (Scale bar: 100 μm.) (iv) High power (×400) light microscopy of anti-von Willebrand immunostaining in day-7 wild-type infarcted heart; (Scale bar: 100 μm.) The vascularity of the infarcted myocardium was increased and multiple vessels containing erythrocytes were observed (black arrows indicate vessels). (Bi) Vascularity of myocardium of wild-type mouse hearts after ligation (filled bars, n = 3-11) or sham surgery (open bars, n = 1-6). Sham-operated animals show a constant vascularity in contrast to CCL animals in which vessel counts increase with time, achieving a maximum at day 7. (Bii) Day-7 hearts from wild-type and 11βHSD1 -/- mice. Ligation (filled bars) in wild-type and 11βHSD1 -/- increased angiogenesis in comparison to sham (open bars) (wild type, n = 6 sham and n = 11 ligations; 11βHSD1-/-, n = 5 sham and n = 10 ligations). Ligations in mice that received RU38486 (dark gray bars, n = 6) induced greater myocardial angiogenesis in comparison to vehicle-treated ligated controls (light gray bars, n = 3). Results are mean ± SEM. #, P < 0.001 versus corresponding sham. ^***, P < 0.001 for differences between wild-type and 11HSD1 -/-.^*, P < 0.02 for differences between coronary artery ligated wild-type mice treated with RU38486 or vehicle. (C) Identification of blood vessels in 7-day-old cutaneous wounds from wild-type mice stained with hematoxylin/eosin (i) or an antibody against von Willebrand factor (ii). (Magnification: ×400; scale bars: 100 μm.)