Glucocorticoids are steroid hormones that have potent anti-inflammatory actions. Endogenous glucocorticoid action is modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyses the interconversion of active glucocorticoids (cortisol, corticosterone) and intrinsically inert forms (cortisone, 11-dehydrocorticosterone). There are 2 isozymes; 11β-HSD type 1 regenerates active glucocorticoids in vivo whereas 11β-HSD type 2 inactivates glucocorticoids. Although 11β-HSD1 is highly expressed in the lung, its role there has been little explored. In this study, the expression and localization of 11β-HSD1 mRNA in lung was confirmed by in situ hybridization. Immunohistochemical staining of mouse lung localized 11β-HSD1 to the cytoplasm of fusiform cells in alveolar walls, in a multivesicular pattern characteristic of interstitial fibroblasts. A lung fibrosis model of inflammation was used to test the role and regulation of 11β-HSD1. The results suggest that levels of 11β-HSD1 mRNA and enzyme were not changed during bleomycin-induced lung inflammation. However, 11β-HSD1-deficient mice showed a more severe inflammatory response than congenic wild-type controls, with greater inflammatory cell infiltration into the lung, and increased levels of HO-1 and iNOS mRNA 14 days following bleomycin installation into lung. Picrosirius red staining of lung sections suggested more collagen deposition in 11β-HSD1-deficient mice than in wild-type controls during the course of the lung inflammatory response. Moreover, whereas naïve 11β-HSD1-deficient mice had significantly lower collagen content in lung (84% of WT levels, p<0.05). 28d after bleomycin there was no significant difference between genotypes (KO having 94% of WT levels, p=0.42) confirming more collagen production in 11β-HSD1-deficient mice following bleomycin. Fibroblasts are critical in the regulation of inflammatory responses and are essential in the model of bleomycin-induced lung injury. Lung fibroblasts may have a different transcriptional regulation of 11β-HSD1 compared to other tissues. In the majority of tissues, 11β-HSD1 can be transcribed from 2 promoters; the P1 promoter is the main promoter used in lung, with other tissues mainly using the P2 promoter. To address the relevance of the P1 promoter in lung and to identify the cell type using the P1 promoter, mouse lungs were collagenase-digested to isolate primary fibroblast and epithelial cells. Isolated lung fibroblasts highly expressed 11β-HSD1, predominantly from the P1 promoter. During passage, primary lung fibroblasts switched promoter usage from P1 to P2. In fibroblast primary culture, treatment with TGF-β for 72h markedly decreased 11β-HSD1 expression to 38% of untreated levels, an effect which was reversed by SB431542, a TGF-β receptor antagonist. Whilst TGF-β reduced levels of mRNA initiating at the P2 promoter, initiation from the P1 promoter was completely repressed. Treatment with TGF-β receptor antagonist increased levels of P1-initiated 11β-HSD1 mRNA by 6.6-fold compared to untreated cells. These data suggest that the switch in 11β-HSD1 promoter usage may be regulated by TGF-β during an inflammatory response. Furthermore, as the P1 and P2 promoters are differentially regulated (e.g. by C/EBPβ, a cytokine-responsive transcription factor), the promoter switch may place 11β-HSD1 under a different transcriptional regulation during inflammation. Taken together, these results suggest that 11β-HSD1 deficiency worsens lung inflammation and results in greater lung fibrosis. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the inflammatory response and shape fibroblast function, limiting subsequent collagen production and fibrosis.

Glucocorticoids can reduce infarct size when given immediately after myocardial infarction (MI) but are detrimental when administration is continued into the post-infarct healing phase. A number of experimental studies have shown that reduction of infarct expansion by enhancing blood supply to the infarct border reduces remodelling and improves heart function post-MI. Previous experiments from this laboratory have shown that mice unable to locally regenerate corticosterone due to deficiency in 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) have an enhanced angiogenic response during myocardial infarct healing that is associated with improved cardiac function. We hypothesized that the enhanced angiogenic response in 11HSD1 knock out (-/-) mice would be preceded by augmented inflammation. Moreover this would be associated with improved cardiac function. This thesis aimed firstly to establish that murine cardiac phenotype was not influenced by 11HSD1 deficiency. 11HSD1-/- and C57Bl6 control mice had comparable cardiac structure and function. 11HSD1 expression was localised to fibroblasts and vascular smooth muscle cells in the myocardium. The second aim of this thesis was to characterise the healing response after MI in 11HSD1-/- mice compared to C57Bl6 mice. Neutrophil infiltration peaked 2 days after MI and was significantly enhanced in the 11HSD1-/- mice relative to C57Bl6 mice, despite comparable infarct size in both groups. This was followed by increased macrophage accumulation in the infarct border. Furthermore, in the 11HSD1-/- mice a greater proportion of macrophages were of the alternatively activated phenotype. Left ventricular expression of pro-angiogenic IL-8, but not VEGF, was increased. Cellular proliferation and vessel density at 7 days were greater in 11HSD1-/- compared to C57Bl6 hearts. This was associated with improved cardiac function 7 days post-MI. The third aim of this thesis was to determine whether the enhancement in vessel density and cardiac function was maintained beyond the initial wound healing phase. 11HSD1-/- mice retained the increased vessel density compared to C57Bl6 mice and these vessels were smooth muscle coated suggesting vessel maturation. This was associated with sustained improvement in cardiac function and modification of the scar characteristics. The final aim of this thesis was to establish whether the effect of the knock out could be recapitulated by administration of a small molecule inhibitor of 11HSD1 after MI. Oral administration of the 11HSD1 inhibitor had no effect on inflammation, angiogenesis and heart function as determined at 7 days post-MI relative to vehicle treated animals. In conclusion, the data confirm the enhancement in vessel density and cardiac function in 11HSD1-/- mice and demonstrate that this was preceded by enhanced inflammation. This was not due to an underlying cardiac phenotype or modification of the infarct size. Increased infiltration of alternatively activated macrophages may have been the source of pro-angiogenic factor, IL-8, which was also increased at the time of angiogenesis. Importantly the enhanced vessel density was retained 4 weeks after MI, these vessels were mature suggesting longevity and the improvement in cardiac function was retained. While pharmacological inhibition did not recapitulate the effect of the knock out this may have been due to route of administration. The data provides compelling evidence that further development and use of small molecule inhibitors of 11HSD1 may be of benefit post-MI.

Inflammation plays a key role in the underlying pathogenesis of obesity and its associated health risks, with increased markers of inflammation evident in both liver and adipose tissue. In parallel, there is dysregulation of glucocorticoid metabolism in obesity, with increased adipose levels of the glucocorticoid-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and increased hepatic levels of 5α-reductase type 1 (5αR1), which catalyses the reduction of glucocorticoids. Both the mechanisms and consequences of this glucocorticoid metabolism dysregulation remain unclear, however, there is evidence that it may be related to inflammation. In vitro studies have demonstrated that pro-inflammatory markers upregulate 11βHSD1 expression in adipocytes, potentially explaining increased expression of this enzyme in obesity. Previous work has also demonstrated that the glucocorticoid metabolites produced by 5αR1 lack the metabolic effects of the parent glucocorticoid, but retain its anti-inflammatory properties, indicating that increased expression of hepatic 5αR1 may serve to dampen down inflammation in the liver. The hypotheses addressed in this thesis are that in obesity, inflammation regulates adipose glucocorticoid metabolism through 11βHSD1, and that hepatic glucocorticoid metabolism regulates the inflammatory state of the liver through 5αR1. The role of inflammation in the regulation of 11βHSD1 was assessed in vivo in mice treated with the anti-inflammatory compound sodium salicylate (salicylate). In diet-induced obese mice, salicylate downregulated 11βHSD1 expression and activity selectively in visceral adipose tissue, alongside improved glucose tolerance, reduced plasma non-esterified fatty acids, and changes in adipose lipid metabolism. 11βHSD1-deficient mice fed a high-fat diet were resistant to the insulin sensitising effects of salicylate treatment. These results indicate a novel role for 11βHSD1 down-regulation in mediating the insulin sensitising effect of anti-inflammatory treatment. The mechanisms underpinning the anti-inflammatory properties of 5α-reduced glucocorticoids were explored in vitro and in vivo. In lipopolysaccharide-stimulated murine macrophages, both 5α-reduced glucocorticoid metabolites tested, namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), suppressed tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) release, although to a lesser extent than corticosterone (B). Similar to B, both 5αDHB and 5α THB suppressed phosphorylation of intra-cellular inflammatory signalling mitogen-activated protein kinases (MAPK) proteins c-Jun N-terminal kinase (JNK) and p38, as well as increasing protein expression of MAPK phosphatase-1 (MKP-1). Treatment of phorbol ester-stimulated HEK293 kidney cells with these 5α-metabolites revealed that 5αDHB suppressed nuclear factor κB (NFκB) and activator protein-1 (AP-1) activation to a similar extent to that of B, whilst 5αTHB increased activation of these pro-inflammatory transcription factors, indicating cell-specific effects of 5αTHB. In conclusion, reduced intra-adipose glucocorticoid regeneration by 11βHSD1 mediates the insulin sensitising effects of salicylate, suggesting that altered glucocorticoid metabolism may reflect altered intra-adipose inflammation in obesity. Furthermore, these data support the concept that this enzyme provides a therapeutic target in obesity-related metabolic disorders. 5α-reduced metabolites of glucocorticoids have similar anti-inflammatory properties to the parent glucocorticoid, indicating that the elevated hepatic levels of 5α-reductase in obesity may be a protective mechanism to limit the adverse metabolic effects of glucocorticoids upon the liver, but maintain the beneficial anti-inflammatory properties. These 5α-reduced glucocorticoid metabolites may provide a potential therapeutic treatment as selective glucocorticoid receptor modulators for inflammatory conditions.

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Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

Background: An important role has been suggested for tissue-specific glucocorticoid metabolism in the development of obesity and its complications. 11ß hydroxysteroid dehydrogenase 1 (11ßHSD1) is an enzyme that catalyzes the interconversion of biologically inactive cortisone to active cortisol, thereby regulating its access to glucocorticoid receptors in target tissues. Indeed, an unfavorable metabolic outcome has been associated with increased 11ßHSD1 gene expression and activity in adipose tissue and liver in humans and rodents. Cortisol is an important regulator of phosphoenolpyruvate carboxykinase (PEPCK) a key enzyme in gluconeogenesis and lipid metabolism. In rodents, overexpression of PEPCK in adipose tissue leads to adiposity and increased fatty acid re-esterification. In human obesity, PEPCK has been positively associated with body fat, total cholesterol levels, and plasma triglycerides. However, few studies have addressed the putative reversibility of peripheral cortisol levels and disturbed fatty acid homeostasis that may accompany weight loss. The aim of this thesis was to investigate alterations in peripheral glucocorticoid metabolism in the context of obesity, and putative modulations of glucocorticoid metabolism in the context of weight changes in humans and rodents. Materials &amp; Methods: 11ßHSD1 expression/activity in different adipose tissue depots and liver, the expression of genes involved in adipogenesis and fatty acid homeostasis, and serum levels of adipose tissue-derived adipokines were investigated in severely obese women before and after surgically induced weight loss. The same parameters were measured in female Sprague-Dawley rats fed on high-fat and control diets. Results: In severely obese women, 11ßHSD1 expression was higher in subcutaneous adipose tissue (SAT), while 11ßHSD1 activity and PEPCK expression were higher in the omental depot. In a multivariate analysis, SAT 11ßHSD1 activity was an independent predictor for central fat accumulation. Hepatic 11ßHSD1 activity and levels of intra-abdominal fat storage correlated negatively, while 11ßHSD1 correlated positively with PEPCK in adipose tissue and liver. Weight loss after gastric bypass surgery was followed by significant and metabolically beneficial reductions in subcutaneous 11ßHSD1 and leptin gene expression, as well as reduced circulating leptin and increased adiponectin levels. In contrast, PEPCK gene expression did not change with weight loss. In rats, a high-fat diet did not affect body weight, but was associated with increased serum leptin and decreased adiponectin levels. Short-term, high-fat diet feeding resulted in the up-regulation of SAT 11ßHSD1 expression, while chronic feeding led to its significant down-regulation (compared with the control diet and short-term, high-fat feeding). Interestingly, hepatic 11ßHSD1 expression was constantly downregulated in rats that were fed a high-fat diet. Conclusions: Severe obesity in women was accompanied by a metabolically adverse increase of 11ßHSD1 in adipose tissue, with a concomitant decrease in the liver. Subcutaneous 11ßHSD1 was an independent predictor for central fat accumulation. As weight loss was followed by significant down-regulation of subcutaneous 11ßHSD1, we suggest that up-regulation of this enzyme was a consequence, rather than a cause of obesity. In rodents, a high-fat diet induced dynamic changes in 11ßHSD1 in SAT and liver, both being down-regulated after chronic high-fat feeding without altered weight. In summary, weight changes and alterations in fat and liver glucocorticoid metabolism are closely linked. Moreover, a high-fat diet significantly influences 11ßHSD1 expression/activity in adipose tissue and liver without affecting body weight.