Mechanisms of actions and roles of 5α-reduced glucocorticoids during inflammation and wound repair

Topical inflammatory diseases are most commonly treated with glucocorticoids, such as hydrocortisone, which have debilitating side effects including a range of systemic metabolic side effects as well as local effects such as to thin the skin and delay wound healing. Safer anti-inflammatory therapies are required and this thesis investigates a novel drug called 5α-tetrahydrocorticosterone (5α-THB) as a safer topical anti-inflammatory treatment. The main foci of this thesis are to assess the effects of 5αTHB on wound repair, as well as to characterise its mechanisms of action. Defective angiogenesis accounts for impaired wound healing brought about by steroids in many cases. 5αTHB suppressed vessel growth in a mouse ex vivo model of angiogenesis, but was less potent in this action than hydrocortisone, suggesting a safer therapeutic profile. To understand the underlying mechanisms, the effect of 5αTHB on gene expression in the mouse aorta during angiogenesis was compared with that of dexamethasone (a selective GR agonist) and hydrocortisone. Whereas dexamethasone and hydrocortisone caused differential expression of genes involved in inflammatory signalling and extracellular matrix remodelling, 5αTHB did not and instead selectively regulated Pecam1, involved in vasculature remodelling. This suggested that 5αTHB suppresses angiogenesis through different mechanisms of action in comparison to dexamethasone, and thus may not act through GR. Supporting this, dexamethasone increased the abundance of GR responsive transcripts (Per1, Hsd11b1, Fkbp51) whereas 5αTHB only increased the abundance of Per1. Furthermore, whereas the GR antagonist RU486 attenuated dexamethasone-regulation of genes, it had no effect on gene regulation by 5αTHB. To assess GR-mediation of 5αTHB effects, model systems were used to investigate whether 5αTHB is able to bind GR, stimulate its nuclear translocation, and initiate changes in its interaction with co regulator peptides. In a competitive binding assay, dexamethasone and hydrocortisone both decreased the fluorescence polarisation of a GR specific ligand, consistent with GR binding. In contrast, 5αTHB only displaced the specific GR ligand at very high concentrations. In terms of nuclear translocation, 5αTHB also did not have an effect on the ratio of GR in the nucleus and cytoplasm (N/C) of A549 cells, suggesting that GR remained predominantly in the cytoplasm after 5αTHB treatment and did not translocate into the nucleus, whereas dexamethasone increased the N/C ratio at three different time points. Likewise, whereas dexamethasone stimulated changes in the interaction between GR and many co regulator peptides, 5αTHB had no effect. Collectively these results from model systems suggest that 5αTHB does not work through the conventional GR mechanism of action. Finally, a hypothesis generating approach was taken in order to gain hints into how 5αTHB may be working. A microarray was performed to compare the effects of 5αTHB and dexamethasone on gene expression in human peripheral blood derived macrophages. Both dexamethasone and 5αTHB were able to cause differential expression of genes in these cells. However unexpectedly, out of the 350 genes regulated by dexamethasone, and the 165 genes regulated by 5αTHB, only 35 genes were commonly regulated by both steroids. This suggested that 5αTHB mainly acts through different mechanisms to dexamethasone also in macrophages. In an enrichment analysis of the differentially expressed genes, whereas the NFκB signalling pathway was the top enriched pathway in genes only regulated by dexamethasone, enriched pathways in genes only regulated by 5αTHB included those related to phagocytosis, the TGF-beta signalling pathway, and Th1-Th2 cell differentiation. This thesis therefore provides evidence to suggest that 5αTHB may provide a safer topical anti-inflammatory steroid, less harmful to wound repair processes. In addition, the mechanisms underlying the action of 5αTHB differ from those of classical GCs, consistent with its reduced side-effect profile. Other potential mechanisms, such as actions through the mineralocorticoid receptor, must now be explored

Safer topical treatment for inflammation using 5α-tetrahydrocorticosterone in mouse models

Use of topical glucocorticoid for inflammatory skin conditions is limited by systemic and local side-effects. This investigation addressed the hypothesis that topical 5α-tetrahydrocorticosterone (5αTHB, a corticosterone metabolite) inhibits dermal inflammation without affecting processes responsible for skin thinning and impaired wound healing. The topical anti-inflammatory properties of 5αTHB were compared with those of corticosterone in C57Bl/6 male mice with irritant dermatitis induced by croton oil, whereas its effects on angiogenesis, inflammation, and collagen deposition were investigated by subcutaneous sponge implantation. 5αTHB decreased dermal swelling and total cell infiltration associated with dermatitis similarly to corticosterone after 24 h, although at a five fold higher dose, but in contrast did not have any effects after 6 h. Pre-treatment with the glucocorticoid receptor antagonist RU486 attenuated the effect of corticosterone on swelling at 24 h, but not that of 5αTHB. After 24 h 5αTHB reduced myeloperoxidase activity (representative of neutrophil infiltration) to a greater extent than corticosterone. At equipotent anti-inflammatory doses 5αTHB suppressed angiogenesis to a limited extent, unlike corticosterone which substantially decreased angiogenesis compared to vehicle. Furthermore, 5αTHB reduced only endothelial cell recruitment in sponges whereas corticosterone also inhibited smooth muscle cell recruitment and decreased transcripts of angiogenic and inflammatory genes. Strikingly, corticosterone, but not 5αTHB, reduced collagen deposition. However, both 5αTHB and corticosterone attenuated macrophage infiltration into sponges. In conclusion, 5αTHB displays the profile of a safer topical anti-inflammatory compound. With limited effects on angiogenesis and extracellular matrix, it is less likely to impair wound healing or cause skin thinning

Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways

<div><p>Glucocorticoids are potent inhibitors of angiogenesis in the rodent <i>in vivo</i> and <i>in vitro</i> but the mechanism by which this occurs has not been determined. Administration of glucocorticoids is used to treat a number of conditions in horses but the angiogenic response of equine vessels to glucocorticoids and, therefore, the potential role of glucocorticoids in pathogenesis and treatment of equine disease, is unknown. This study addressed the hypothesis that glucocorticoids would be angiostatic both in equine and murine blood vessels.The mouse aortic ring model of angiogenesis was adapted to assess the effects of cortisol in equine vessels. Vessel rings were cultured under basal conditions or exposed to: foetal bovine serum (FBS; 3%); cortisol (600 nM), cortisol (600nM) plus FBS (3%), cortisol (600nM) plus either the glucocorticoid receptor antagonist RU486 or the mineralocorticoid receptor antagonist spironolactone. In murine aortae cortisol inhibited and FBS stimulated new vessel growth. In contrast, in equine blood vessels FBS alone had no effect but cortisol alone, or in combination with FBS, dramatically increased new vessel growth compared with controls. This effect was blocked by glucocorticoid receptor antagonism but not by mineralocorticoid antagonism. The transcriptomes of murine and equine angiogenesis demonstrated cortisol-induced down-regulation of inflammatory pathways in both species but up-regulation of pro-angiogenic pathways selectively in the horse. Genes up-regulated in the horse and down-regulated in mice were associated with the extracellular matrix. These data call into question our understanding of glucocorticoids as angiostatic in every species and may be of clinical relevance in the horse.</p></div

Cortisol inhibits angiogenesis in murine aortae.

<p>New vessel outgrowths from murine aortae (C57BL/6J, male, 8 weeks of age, n = 10) in the presence of DMEM, foetal bovine serum (FBS), cortisol, FBS+ cortisol, cortisol + RU486, or cortisol + spironolactone. Data are mean ± SEM and were analysed by one-way ANOVA and Dunnett’s post-hoc test at each time point. * P<0.05in comparison to DMEM.</p

Cortisol stimulates angiogenesis in equine vessels.

<p>(a) Light microscopy images of new vessel outgrowths [i], which stained strongly (brown) for CD31 [ii], indicating they are likely to be predominantly endothelial in nature (Scale 0.2mm). (b) Light microscopy images of equine laminar vessel sections after incubation (5 days) with DMEM [i], foetal bovine serum [ii], cortisol [iii], or FBS with cortisol [iv]. These demonstrate the stimulatory effect of cortisol on new vessel growth from equine vessels. (c) New vessel outgrowths from laminar (n = 10) [i] and facial skin vessels (n = 10) [ii] of healthy horses were quantified in the presence of DMEM, foetal bovine serum (FBS), cortisol, FBS+ cortisol, cortisol + RU486, or cortisol + spironolactone. Data are mean ± SEM for (n) horses) and were analysed by one-way ANOVA and Dunnett’s post-hoc test at each time point. * P<0.05 in comparison to DMEM.</p

GO term enrichment analysis comparing murine samples incubated with FBS (control) with those incubated with cortisol.

<p>The top 8 up-regulated and down-regulated pathways are shown.</p

Genes identified by next-generation sequencing that were up-regulated in the horse by cortisol and down-regulated in the mouse.

<p>Genes identified by next-generation sequencing that were up-regulated in the horse by cortisol and down-regulated in the mouse.</p

Genes identified by next-generation sequencing that were down-regulated in the horse by cortisol and up-regulated in the mouse.

<p>Genes identified by next-generation sequencing that were down-regulated in the horse by cortisol and up-regulated in the mouse.</p

KEGG enrichment analysis of pathways up- or down-regulated in equine laminar vessels in response to cortisol.

<p>Thirty six KEGG pathways were identified as significantly enriched. Eight were up-regulated (red) and 28 were down-regulated (blue).</p