Abstract
BackgroundGlucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. However, the mechanisms through which glucocorticoids inhibit growth of new blood vessels have not been established. This study addresses the hypothesis that physiological levels of glucocorticoids inhibit angiogenesis by directly preventing tube formation by endothelial cells.Methodology/Principal FindingsCultured human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells were used to determine the influence of glucocorticoids on tube-like structure (TLS) formation, and on cellular proliferation (5-bromo-2′-deoxyuridine (BrdU) incorporation), viability (ATP production) and migration (Boyden chambers). Dexamethasone or cortisol (at physiological concentrations) inhibited both basal and prostaglandin F2α (PGF2α)-induced and vascular endothelial growth factor (VEGF) stimulated TLS formation in endothelial cells (ECs) cultured on Matrigel, effects which were blocked with the glucocorticoid receptor antagonist RU38486. Glucocorticoids had no effect on EC viability, migration or proliferation. Time-lapse imaging showed that cortisol blocked VEGF-stimulated cytoskeletal reorganisation and initialisation of tube formation. Real time PCR suggested that increased expression of thrombospodin-1 contributed to glucocorticoid-mediated inhibition of TLS formation.Conclusions/SignificanceWe conclude that glucocorticoids interact directly with glucocorticoid receptors on vascular ECs to inhibit TLS formation. This action, which was conserved in ECs from two distinct vascular territories, was due to alterations in cell morphology rather than inhibition of EC viability, migration or proliferation and may be mediated in part by induction of thrombospodin-1. These findings provide important insights into the anti-angiogenic action of endogenous glucocorticoids in health and disease.
Highlights
The well-documented ability of glucocorticoids to inhibit angiogenesis [1] is exploited clinically for the reduction of proliferating capillary haemangiomas [2] and may have potential in treatment of some cancers
tube-like structure (TLS) formed by human umbilical vein ECs (HUVECs) retain an endothelial cells (ECs) phenotype HUVECs cultured on plastic retained a typical cobblestone appearance
Data represent mean6standard error of mean (SEM) (n = 3–6, each condition performed in triplicate) and were analysed using one-way analysis of variance (ANOVA) and Dunnett’s post hoc test (*p,0.05, **p,0.01). doi:10.1371/journal.pone.0014476.g002. This investigation addressed the hypothesis that the potent antiangiogenic action of glucocorticoids is due to prevention of tube formation by endothelial cells. These results show that glucocorticoids do induce direct, glucocorticoid receptor (GR)-mediated inhibition of tube formation by primary human ECs
Summary
The well-documented ability of glucocorticoids to inhibit angiogenesis [1] is exploited clinically for the reduction of proliferating capillary haemangiomas [2] and may have potential in treatment of some cancers. There is increasing evidence that endogenous glucocorticoids contribute to regulation of new vessel formation (reviewed in [4]). Suppression of angiogenesis may contribute to impaired wound healing in glucocorticoid excess [5] whilst prereceptor regulation of glucocorticoid concentrations in target tissues (by the isozymes of 11b-hydroxysteroid dehydrogenase; 11b-HSD) has been linked to both physiological and pathophysiological angiogenesis. Generation of glucocorticoids by 11bHSD type 1 has been shown to inhibit recovery from cutaneous wounds and myocardial infarction [7], and increase age-related bone fragility [8] by suppressing angiogenesis. Glucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. The mechanisms through which glucocorticoids inhibit growth of new blood vessels have not been established. This study addresses the hypothesis that physiological levels of glucocorticoids inhibit angiogenesis by directly preventing tube formation by endothelial cells
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