Linsay J Macdonald's research while affiliated with The University of Edinburgh and other places

Exogenous glucocorticoids inhibit neointimal proliferation in animals. We aimed to test the hypothesis that endogenous glucocorticoids influence neointimal proliferation; this may be mediated by effects on systemic risk factors or locally in vessels and modulated by either adrenal secretion or enzymes expressed in vessels that mediate local inactivation [11β-hydroxysteroid dehydrogenase type II (11β-HSD2) in endothelium] or regeneration [11β-hydroxysteroid dehydrogenase type I (11β-HSD1) in smooth muscle] of glucocorticoids. Femoral artery wire angioplasty was conducted in C57BL/6J, Apo-E(-/-), 11β-HSD1(-/-), Apo-E, 11β-HSD1(-/-) (double knockout), and 11β-HSD2(-/-) mice after glucocorticoid administration, adrenalectomy, glucocorticoid or mineralocorticoid receptor antagonism, or selective 11β-HSD1 inhibition. In C57BL/6J mice, neointimal proliferation was reduced by systemic or local glucocorticoid administration, unaffected by adrenalectomy, reduced by the mineralocorticoid receptor antagonist eplerenone, and increased by the glucocorticoid receptor antagonist RU38486. 11β-HSD2 deletion had no effect on neointimal proliferation, with or without eplerenone. 11β-HSD1 inhibition or deletion had no effect in chow-fed C57BL/6J mice but reduced neointimal proliferation in Apo-E(-/-) mice on Western diet. Reductions in neointimal size were accompanied by reduced macrophage and increased collagen content. We conclude that pharmacological administration of glucocorticoid receptor agonists or of mineralocorticoid receptor antagonists may be useful in reducing neointimal proliferation. Endogenous corticosteroids induce beneficial glucocorticoid receptor activation and adverse mineralocorticoid receptor activation. However, manipulation of glucocorticoid metabolism has beneficial effects only in mice with exaggerated systemic risk factors, suggesting effects mediated primarily in liver and adipose rather than intravascular glucocorticoid signaling. Reducing glucocorticoid action with 11β-HSD1 inhibitors that are being developed for type 2 diabetes appears not to risk enhanced neointimal proliferation.

Antagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or D-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9-10) neurones where GluN2B expression predominates. In older cultures (DIV 15-18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility.

Rationale Glucocorticoid administration increases cardiovascular risk but inhibits neointimal proliferation in animal models. Glucocorticoid activity in target tissues is regulated by the isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type-1 regenerates active glucocorticoids and type-2 inactivates glucocorticoids. Both 11β-HSD isozymes are expressed in vessel wall as well as in liver and adipose tissue; they may modulate vascular remodelling by acting locally or by influencing systemic risk factors. This study investigated effects of these isozymes on post-angioplasty neointimal proliferation. Methodology Wire angioplasty was conducted in femoral arteries of C57Bl/6J, Apo-E knockout, 11β-HSD1 knockout, Apo-E/11β-HSD1 double-knockout and 11β-HSD2 knockout mice. Effects of pharmacological inhibition of 11β-HSD1 (Compound 544, 30 mg/kg/day) were also studied. Vascular lesions were assessed using novel optical projection tomography and standard histology. Results 11β-HSD1 deletion or inhibition resulted in ∼35% reduction in neointimal volume with corresponding increase in lumen size in western-diet fed Apo-E knockout mice but had no significant effect in chow-fed C57Bl/6J mice. Reduced neointimal proliferation was associated with reduction in weight gain, insulin resistance, systolic blood pressure and macrophage content of neointimal lesions. 11β-HSD2 knockout mice had substantially higher blood pressure than C57Bl/6J mice. Neointimal proliferation, however, was similar in both groups, albeit 11β-HSD2 knockout mice having higher neointimal macrophage content. Conclusion 11β-HSD1 inhibition ameliorates multiple cardiovascular risk factors and reduces neointimal proliferation only in mice with exaggerated systemic risk factors, suggesting effects mediated primarily in liver and adipose. Any local amplification (with disruption of 11β-HSD2) or reduction (with disruption of 11β-HSD1) in glucocorticoid concentration within the vessel wall is likely to be insufficient to overcome the effect of systemic risk factors on neointimal proliferation.

11beta-hydroxysteroid dehydrogenases (11betaHSDs) catalyze interconversion of 11-hydroxy-glucocorticoids with inactive 11-keto metabolites. In blood vessel walls, loss of 11betaHSD1 is thought to reduce local glucocorticoid concentrations, reducing the progression of atheroma and enhancing angiogenesis. Conversely, on the basis that 11betaHSD1 is up-regulated approximately 5-fold by inflammatory cytokines in cultured human vascular smooth muscle cells, it has been proposed that increased 11betaHSD1 during vascular inflammation provides negative feedback suppression of inflammation. We aimed to determine whether inflammation and injury selectively up-regulate 11betaHSD1 reductase activity in vitro and in vivo in intact vascular tissue in mice. In isolated mouse aortae and femoral arteries, reductase activity (converting 11-dehydrocorticosterone to corticosterone) was approximately 10-fold higher than dehydrogenase activity and was entirely accounted for by 11betaHSD1 because it was abolished in vessels from 11betaHSD1(-/-) mice. Although 11betaHSD1 activity was up-regulated by proinflammatory cytokines in cultured murine aortic smooth muscle cells, no such effect was evident in intact aortic rings in vitro. Moreover, after systemic inflammation induced by ip lipopolysaccharide injection, there was only a modest (18%) increase in 11beta-reductase activity in the aorta and no increase in the perfused hindlimb. Furthermore, in femoral arteries in which neointimal proliferation was induced by intraluminal injury, there was no change in basal 11betaHSD1 activity or the sensitivity of 11betaHSD1 to cytokine up-regulation. We conclude that increased generation of glucocorticoids by 11betaHSD1 in the murine vessel wall is unlikely to contribute to feedback regulation of inflammation.

The ability of glucocorticoids to directly alter arterial function, structure and the inflammatory response to vascular injury may contribute to their well-established link with the development of cardiovascular disease. Recent studies have emphasised the importance of tissue-specific regulation of glucocorticoid availability by the 11 beta-hydroxysteroid dehydrogenase (11HSD) isozymes, which inter-convert active glucocorticoids and their inactive metabolites. The expression of both type 1 and type 2 11HSDs in the arterial wall suggests that prereceptor metabolism of glucocorticoids may have a direct impact on vascular physiology. Indeed there is evidence that 11HSDs influence glucocorticoid-mediated changes in vascular contractility, vascular structure, the inflammatory response to injury and the growth of new blood vessels. Hence, inhibition of 11HSD isozymes may provide a novel therapeutic target in vascular disease.

Metabolism of glucocorticoids to A-ring-reduced dihydro- and tetrahydro-derivatives by means of hepatic 5α- and 5β-reductases has long been regarded as a pathway of irreversible inactivation. However, 5α-reduced metabolites of other steroids, e.g. testosterone and aldosterone, have significant biological activity. We investigated whether 5α-reduced metabolites of corticosterone are glucocorticoid receptor (GR) agonists. Corticosterone, 5α-tetrahydrocorticosterone (5αTHB), and 5α-dihydrocorticosterone (5αDHB) were similarly effective in displacing tritiated dexamethasone from binding sites in hepatocytes, whereas 5β-reduced metabolites were less effective in binding. 5αTHB had glucocorticoid receptor agonist effects in vitro and in vivo. After transient co-transfection of hGR and a murine mammary tumor virus-luciferase reporter into HeLa cells, 5αTHB was active to a comparable extent as corticosterone (28-fold versus 37-fold induction, respectively, at 1 μm) and additive to the effect of corticosterone. 5β-Reduced metabolites did not activate GR. In H4IIE hepatoma cells, both 5αTHB and corticosterone induced mRNA expression of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (6.0-versus 10.1-fold and 3.5-versus 3.9-fold at 1 μm, respectively), an effect that was inhibited by RU486. To assess in vivo glucocorticoid activity, suppression of plasma ACTH was demonstrated in adrenalectomized rats after intraperitoneal administration of vehicle (ACTH trough 80.2 pm), corticosterone (5 mg/kg; 22 pm, p < 0.001) or 5αTHB (5 mg/kg; 51.3 pm, p < 0.005). Similar endogenous concentrations of corticosterone and 5αTHB were detected in rat liver homogenates by gas chromatography mass spectrometry. We conclude that 5α-reduced glucocorticoids bind to and activate GR. Transcription of glucocorticoid-regulated genes in tissues that express 5α-reductases will thus be influenced by intracellular levels of both corticosterone and its 5α-reduced metabolites.