No description available.

No description available.

Atherosclerotic and restenotic lesions develop as a result of an excess inflammatory response to vascular injury. Glucocorticoid hormones have widely-recognised anti-inflammatory and anti-proliferative properties which appear to make them ideal candidates for inhibition of vascular lesion development. Indeed, administration of glucocorticoids to experimental animals does inhibit the growth of vascular lesions in some models. In addition, glucocorticoids are currently being trialled clinically as anti-restenotic agents. However, glucocorticoid excess in patients, either as a result of Cushing’s syndrome or chronic steroid therapy, is associated with enhanced CVD risk. Therefore, the effects of glucocorticoids on vascular lesion development remain imperfectly understood. The overall objective of these studies was to explore the influence of endogenous and exogenous glucocorticoids on vascular lesion development using murine models of atherosclerosis (ApoE-/- mice fed a “western” diet) and neointimal hyperplasia (wireinduced femoral artery injury). The work described in this thesis addresses the hypothesis that glucocorticoids are pro-atherogenic, yet anti-restenotic. Mice were bilaterally adrenalectomised to investigate the role of endogenous glucocorticoids on vascular lesion development. Removal of the adrenal glands had no influence on atherogenesis or neointima development. The influence of exogenous glucocorticoids on lesion development was assessed by orally administering dexamethasone (0.1 or 0.8mg/kg/day). Atherosclerotic lesion burden was augmented by dexamethasone administration. Conversely, fibro-proliferative neointimal proliferation was inhibited by dexamethasone. However, this effect was obscured by thrombotic lesion development. It was proposed that this thrombotic effect is attributable to increased plasminogen activator inhibitor-1 (PAI-1), which was tested using PAI-1 deficient mice. Although PAI-1 was found to mediate the systemic pro-thrombotic effect of dexamethasone, it is not required for the enhanced development of thrombotic lesions at the site of intra-luminal injury. These results suggest that physiological levels of endogenous glucocorticoids play a limited role in vascular lesion development. Conversely, although exogenous glucocorticoids inhibit fibro-proliferative intimal hyperplasia, they appear to have significant detrimental influences on lesion development, augmenting atherosclerosis and inducing thrombotic neointimal lesion formation following vascular injury. Further research is therefore required to improve the cardiovascular outcome of patients requiring glucocorticoid therapy and for the use of glucocorticoids as antirestenotic agents.

Abstract not available

Background: Although glucocorticoids are the most effective treatment for asthma, some patients show a poor response. In such patients with steroid resistant asthma, this has been ascribed to altered glucocorticoid receptor (GR) ligand-binding affinity induced by IL-2 combined with IL-4 or IL-13 alone- all of which can also modulate glucocorticoid function in vitro. Objective: We sought to assess the ligand-binding affinity in a distinct group of oral steroid-dependent asthmatic subjects and examine the mechanisms by which IL-2 and IL-4 (or IL-13) modify the ligand-binding affinity of the GR. Methods: Using dexamethasone-binding assays, we examined PBMCs ex vivo from healthy subjects, subjects with controlled asthma, and oral steroiddependent subjects with severe asthma. In addition, IL-2 and IL-4 were used to alter GR affinity in vitro. We used mediators or inhibitors of signal transduction to investigate the mechanisms of resistance. We also determined cytokine production of PBMC's by means of ELISA. Results: GR ligand-binding affinity was significantly reduced in the nucleus but not in the cytoplasm of oral steroid-dependent asthmatic subjects compared with that seen in steroid-sensitive and healthy subjects (dissociation constant, 41.37 ± 17.83 vs. 25.36 ± 2.63 nmol/L vs. 9.40 ± 4.01 nmol/L, respectively [p<.05 for both in comparison to normals] ). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 co-treatment and was blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. PBMC's rendered resistant in vitro demonstrated lower IL-10 and increased GM-CSF production following LPS or PMA & PHA stimulation compared to cells with normal GR affinity. Resistant cells also showed reduced dexamethasone repression of LPSstimulated IL-10 release. These effects were also reversed by SB203580. Inhibition of the ERK MAPK pathway by PD098059 (10 mol/L), phosphoinositol 3 kinase by wortmannin (5 nmol/L) or treatment with IL-10 (10 ng/mL) failed to modulate the effect of IL-2 and IL-4 on receptor affinity. Ro318220 (10 nmol/L), a specific protein kinase C inhibitor and theophylline, similarly, had no effect on affinity. Conclusion: GR ligand binding affinity is tiered; compared to normal subjects; steroid responsive asthmatics have a mild reduction in ligand binding whereas oral steroid dependent asthmatics have greater reductions. When mononuclear cells are rendered resistant in vitro, cytokine production (low IL-10 and high GM-CSF) favours a pro-inflammatory state. Our data do not support the ERK MAPK, phosphoinositol 3 kinase, protein kinase C pathways in steroid resistance. Treatment with IL-10 and theophylline also failed to modulate the effect of IL-2 and IL-4 on receptor affinity. However, P38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and re-establishing the beneficial effects of glucocorticoids in patients with severe asthma. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves : 138-152). Also available on the Internet.

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.

It is well established that atherosclerosis, an inflammatory response to chronic injury in the blood vessel wall, plays a leading role in the development and progression of cardiovascular disease. Mineralocorticoid receptor (MR) over-activation has been implicated in atherosclerosis. In mineralocorticoid-target tissues, 11β- Hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates glucocorticoids, conferring aldosterone specificity upon the normally unselective MR. Recent evidence suggests that 11β-HSD2 may also afford protection of MR in the cells of the vasculature, providing possible mechanisms by which MR activation may directly promote atherosclerosis. Consistent with this, Apoe-/-/11β-HSD2-/- double knockout (DKO) mice show accelerated atheroma development. The present thesis tested the hypothesis that inactivation of 11β-HSD2, allowing inappropriate activation of MR in cells of the vasculature, accelerates atherogenesis through promotion of a pro-inflammatory environment with increased endothelial cell expression of adhesion molecules and subsequent macrophage infiltration into plaques. DKO mice received either the MR antagonist eplerenone (200mg/kg/day) or vehicle in normal chow diet from 2 months of age for 12 weeks. Eplerenone significantly decreased atherosclerotic burden in brachiocephalic arteries of DKO mice, an effect that was accompanied by alterations in the cellular composition of plaques such that a more stable collagen- and smooth muscle cell- rich plaque was formed. Eplerenone treatment was also associated with a reduction in vascular inflammation as demonstrated by a significant reduction in macrophage infiltration into DKO plaques. The accelerated atherogenesis in DKO mice was clearly evident by 3 months of age, a time point at which Apoe-/- mice were completely lesion free. By 6 months, some Apoe-/- mice had developed lesions whilst all DKO mice at this age showed much larger plaques. Compared to Apoe-/- mice, the cellular composition of DKO plaques was altered favouring vulnerability and inflammation, with increased macrophage and lipid content and decreased collagen content. To investigate the possible underlying mechanisms responsible for increased inflammatory cell content, the expression of vascular cell adhesion molecule 1 (VCAM-1) was compared in DKO and Apoe-/- brachiocephalic arteries. VCAM-1 immunostaining was significantly greater on the endothelial cells of DKO arteries at 3 months compared to age-matched Apoe-/- mice. At 6 months, DKO and Apoe-/- mice had similar expression of VCAM-1. Finally, mouse aortic endothelial cells (MAECs) were used to investigate the mechanism of adhesion molecule up-regulation in the absence of 11β-HSD2. Both aldosterone and TNF-α, included as a positive control, dramatically increased VCAM-1 expression in MAECs. Spironolactone pre-treatment blocked the effect of aldosterone, suggesting an MR-mediated mechanism. Corticosterone alone had no effect on VCAM-1 expression. However, inhibition of 11β-HSD2 by pre-treatment with glycyrrhetinic acid allowed corticosterone to induce a significant increase in the number of VCAM-1-stained MAECs, demonstrating functional expression of 11β- HSD2 in MAECs. Consistent with 11β-HSD2 involvement, VCAM-1 up-regulation by corticosterone in the presence of glycyrrhetinic acid was reversed by blockade of MR with spironolactone. In conclusion, loss of 11β-HSD2 activity leading to inappropriate activation of MR in atherosclerotic mice promotes plaque vulnerability and increases vascular infiltration of macrophages which accelerates plaque growth, possibly through enhanced MR- mediated endothelial cell expression of VCAM-1.