Recent epidemiological data have implicated prenatal events in the development of cardiovascular disorders. Thus low birth weight strongly predicts the later occurrence of hypertension, type II diabetes mellitus, syndrome X and deaths from ischaemic heart disease. The mechanism linking prenatal events and later disease is not clear, although maternal malnutrition has been advocated. We have advanced the hypothesis that glucocorticoids might be important as they retard foetal growth and programme offspring hypertension in rats. The foetus has been thought to be protected from the 2-10 times higher maternal glucocorticoid levels by the placental enzyme 11B-hydroxysteroid dehydrogenase (11B-HSD), which is present in many tissues and in humans catalyses the conversion of the active glucocorticoid cortisol to inert cortisone (corticosterone to 11-dehydrocorticosterone in rats). The precise role of 11B-HSD as a barrier to maternal glucocorticoids during prenatal life has not been fully characterised. The role of 11B-HSD in controlling prenatal glucocorticoid exposure in humans and animals has thus been examined. Two isoforms of 11B-HSD exist, type 1, a widespread NADP dependent reversible enzyme and type 2, a high affinity NAD dependent dehydrogenase found mainly in placenta and kidney. 11B-HSD was found in abundance in the ovary and placenta. The main site of immunohistochemical staining and expression of mRNA (11B-HSD-1) in the rat ovary was in the oocyte. 11B-HSD was oxidative, inactivating corticosterone. In both rat placenta in-vitro (11B-HSD-2), and human placenta in-vitro and ex-vivo (11B-HSD-2) the bioactivity was also predominantly oxidative. The lowest placental enzyme activity at term (and hence the greatest foetal glucocorticoid exposure) was found in the smallest rats with the largest placentas, i.e. those in human studies who would be predicted to develop the highest adult blood pressures (birth weight vs. placental 11B-HSD activity: n = 56; r = 0.46; p < 0.0005). A method to examine 11B-HSD function in fresh intact human placentas was developed (ex-vivo dual circuit cotyledon perfusion) which allows close approximation to the in-vivo situation. The majority of cortisol, from low to high nanomolar concentrations, infused through the maternal circulation was metabolised to inert cortisone by the time it reached the foetal circulation, although considerable individual variation was observed. 118-HSD was the only significant contributor to placental cortisol metabolism at physiological maternal concentrations and inhibition of 118-HSD with either the liquorice constituent glycyrrhetinic acid or its hemi-succinate, carbenoxolone, resulted in abolition of the glucocorticoid barrier, allowing maternally administered cortisol to pass unmetabolised through the placenta. In a prospective study, on 16 normal primiparous women whose placentas were studied with this technique, a positive and significant correlation was found between the effectiveness of 118-HSD and offspring birth weight (r = 0. 67; p < 0. 005). The relationship between placental 118-HSD effectiveness in-vivo and term cord blood osteocalcin (a sensitive marker of glucocorticoid exposure) was prospectively examined in 19 women. Cord blood levels of the bone specific protein osteocalcin were determined with radioimmunoassay. The lowest cord blood osteocalcin levels were found in the foetuses whose placental 118-HSD barrier function was poorest (r = 0.58; p < 0.02), (and had presumably had the greatest glucocorticoid exposure), suggesting that term cord blood osteocalcin levels might be a useful predictor of hypertension, ischaemic heart disease and possibly metabolic bone disease. The findings presented in this thesis represent direct evidence that 118-HSD is the barrier to maternal glucocorticoids, its effectiveness correlating with foetal growth in rats (in-vitro), in humans (ex-vivo), and in-vivo with human cord blood osteocalcin levels (osteocalcin may be a marker of glucocorticoid exposure). In the light of studies on pregnant rats in which administration of exogenous glucocorticoids or 118-HSD inhibitors reduces birth weight and programmes hypertension in the offspring, it is reasonable to propose that increased foetal glucocorticoid exposure consequent upon attenuated placental 118-HSD function may play a role in intrauterine programming of later hypertension.

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.

Abstract The biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens. In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence. In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2. The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer.

Corticosterone in foetal tissues after injection of the mother with ¹⁴C-corticosterone was determined by acetylation. with ³H-acetic anhydride and crystallization to constant specific activity. The corticosterone content of whole foetal tissue varied between gestational days 13 and 17 from 641 to 300 ng/g respectively. The specific activity of foetal hormone recovered remained essentially constant; after a 15-minute pulse this was as much as one-fourth that of maternal hormone. However, placenta, head and liver showed distinctly different patterns of metabolism, which changed greatly during this time in head and liver, with a decrease in the conversion of corticosterone to 11-dehydrocorticosterone and a rise in foetal liver 113-hydroxysteroid:NADP oxidoreductase activity. This mitochondrial enzyme, Km=33yM, pH optimum 6, which reduces the 11-dehydro metabolite to the biologically active 116-OH compound, increased sharply, raising the relative amount of the latter in foetal tissues from 15 to 91% during this period. One day after removal of maternal adrenals, foetal corticosterone was normal and maternal levels close to normal, indicating ability of foetal adrenals to function. Maternal hormone, however, crossed to the foetus readily and it was considered most likely that, normally, the maternal source predominates. Regardless of origin, foetal or maternal, however, the hormone is maintained in different foetal tissues in a distinct and different manner. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21719号 / 薬科博第110号 / 新制||薬科||12(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Intra-adipose cortisol is derived from the systemic circulation via the hypothalamic-pituitaryadrenal axis (HPAA) and generated locally through conversion of inactive cortisone to cortisol by the intra-cellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). This thesis addresses the relative contributions of the HPAA and adipose tissue 11βHSD1 to the adipose tissue glucocorticoid pool and describes development and validation of a novel stable isotope tracer, 1,2 [2H]2-cortisone (d2-cortisone), to measure 11βHSD1- dehydrogenase activity in adipose tissue and skeletal muscle in vivo. In otherwise healthy females (n=6) undergoing hysterectomy for a benign indication, an intravenous infusion of d4-cortisol was administered and subcutaneous and omental adipose tissue biopsies were obtained along with concomitant peripheral venous blood, to measure the rate of exchange of cortisol between plasma and adipose tissue for comparison with rates of intra-cellular cortisol generation by 11βHSD1. Cortisol concentrations and enrichment with d4-cortisol were lower in adipose tissue than in plasma. The rate of accumulation of d4-cortisol in adipose tissue depots was ~0.5nmol/kg/h despite the infusion contributing 1.9μmol/h d4-cortisol into the circulation, and the proportion of the intra-adipose cortisol pool replaced each hour was ~10%. The contribution of 11βHSD1 to this turnover could not be quantified since very little substrate d3-cortisone accumulated in adipose during infusion. Method development for d2-cortisone included optimising LC-MS/MS conditions, confirming that d2-cortisone was a substrate for human 11βHSD1 and that no significant primary isotope effect existed. The pharmacokinetics of d2-cortisone were assessed in vivo in healthy male volunteers (n=3). The method was validated by measuring whole body cortisone production in healthy volunteers (n=3) before and after eating liquorice which resulted in a ~50% fall in cortisone production. 11βHSD1-dehydrogenase activity was measured in adipose tissue and skeletal muscle in healthy volunteers (n=6) using d2- cortisone and substantial 11β-dehydrogenase activity was present in both tissues (~1.5-fold higher 11β-dehydrogenase activity than 11β-reductase activity in adipose tissue and approximately equal 11β-reductase and 11β-dehydrogenase activity in skeletal muscle). 11βHSD1-reductase activity was also assessed using a 9,11,12,12 [2H]4-cortisol infusion (d4-cortisol). Skeletal muscle and adipose tissue displayed 11β-reductase activity. In adipose tissue this activity was of a similar magnitude to previous reports. Insulin increased whole body 11β-reductase activity, but did not switch 11βHSD1 direction in muscle or adipose tissue, indicating the predominant effect of insulin may be on hepatic 11βHSD1. Therefore, turnover of the intra-adipose tissue glucocorticoid pool is slow and it is unlikely that rapid acute fluctuations in circulating cortisol are reflected in adipose tissue, although this has not been confirmed under normal physiological conditions. Secondly, 11βHSD1 may be bidirectional in human subcutaneous adipose tissue and skeletal muscle in vivo, and insulin does not regulate the balance of activities. However, in this study blood sampling occurred from blood vessels which express 11βHSD2, and thus some of the measured dehydrogenase activity in this study may reflect endothelial 11βHSD2 activity. Together these findings further our understanding of adipose tissue cortisol physiology in health, suggesting that 11βHSD1 may play a relatively important role in modulating activation of glucocorticoid receptors in adipose tissue, and that dysregulation or inhibition of 11βHSD1 may affect cortisol inactivation as well as regeneration.

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.

Epithelial Ovarian Cancer (EOC) kills more women annually in the United Kingdom than any other gynaecological cancer. Survival rates for women diagnosed with EOC have not improved over the past 30 years, due to the often advanced stage at presentation, where widespread intra-peritoneal dissemination has occurred. The natural history of the disease remains uncertain but the ovarian surface epithelium (OSE) is a strong candidate for the tissue of origin. The OSE undergoes cyclical damage and repair in women of reproductive age following ovulation, which can be considered an acute inflammatory event. Factors that prevent ovulation (pregnancy, breastfeeding and contraceptive pill use) also protect against the development of EOC. Previously published data show that the OSE is able to upregulate the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) in response to inflammation, the enzyme responsible for converting inactive cortisone to anti-inflammatory cortisol. This thesis hypothesises that 11βHSD isozymes are deregulated in ovarian cancer; that the peritoneal surface epithelium (PSE) is indistinguishable from the OSE in its response to inflammation and should be considered a potential source of some “ovarian cancers”; and finally that the expression of the tumour suppressor gene OPCML (OPioid binding Cell adhesion Molecule-Like) is altered by inflammation. These hypotheses were examined at three levels. Firstly, primary cultures of EOC were established, and glucocorticoid metabolism and the response to inflammation was compared to normal OSE. Results from these investigations reveal that the11βHSD1 response to IL-1α stimulation is impaired in EOC compared to normal OSE at the mRNA level but there is no significant difference when 11βHSD1 enzyme activity is measured in these tissues. When basal levels of 11βHSD1, 11βHSD2 and COX2 are compared amongst untreated samples of EOC and OSE, there was a significant correlation between 11βHSD1 and COX2 mRNA expression (P<0.001). 11βHSD2 mRNA expression was significantly higher in the EOC specimens compared to OSE (P<0.05). Secondly the response to inflammation was compared in primary cultures of human peritoneal surface epithelial (PSE) cells and OSE. The data suggest that the mRNA response to inflammation was similar in OSE and PSE, but that the 11βHSD1 enzyme activity was reduced in PSE (P<0.05), which may result in differences in tissue healing. Finally, the effect of inflammation on the expression of the ovarian cancer associated tumour suppressor gene (TSG), OPCML (OPioid binding Cell adhesion Molecule-Like) and the other members of the IgLON family, was examined in OSE. These results suggest that OPCML mRNA expression can be induced by IL-1α, an effect that is inhibited by cortisol.

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