5α-Tetrahydrocorticosterone: A topical anti-inflammatory glucocorticoid with an improved therapeutic index in a murine model of dermatitis.

BACKGROUND AND PURPOSE: Glucocorticoids are powerful anti-inflammatory drugs, but are associated with many side-effects. Topical application in atopic dermatitis leads to skin thinning, metabolic changes, and adrenal suppression. 5α-Tetrahydrocorticosterone (5αTHB) is a potential selective anti-inflammatory with reduced metabolic effects. Here, the efficacy and side-effect profile of 5αTHB were compared with hydrocortisone in preclinical models of irritant dermatitis. EXPERIMENTAL APPROACH: Acute irritant dermatitis was invoked in ear skin of male C57BL/6 mice with a single topical application of croton oil. Inflammation was assessed as oedema via ear weight following treatment with 5αTHB and hydrocortisone. Side-effects of 5αTHB and hydrocortisone were assessed following chronic topical steroid treatment (28 days) to non-irritated skin. Skin thinning was quantified longitudinally by caliper measurements and summarily by qPCR for transcripts for genes involved in extracellular matrix homeostasis; systemic effects of topical steroid administration also were assessed. Clearance of 5αTHB and hydrocortisone were measured following intravenous and oral administration. KEY RESULTS: 5αTHB suppressed ear swelling in mice, with ED50 similar to hydrocortisone (23 µg vs. 13 µg). Chronic application of 5αTHB did not cause skin thinning, adrenal atrophy, weight loss, thymic involution, or raised insulin levels, all of which were observed with topical hydrocortisone. Transcripts for genes involved in collagen synthesis and stability were adversely affected by all doses of hydrocortisone, but only by the highest dose of 5αTHB (8× ED50 ). 5αTHB was rapidly cleared from the systemic circulation. CONCLUSIONS AND IMPLICATIONS: Topical 5αTHB has potential to treat inflammatory skin conditions, particularly in areas of delicate skin.

Comparison of mechanisms of angiostasis caused by the anti-inflammatory steroid 5α-tetrahydrocorticosterone versus conventional glucocorticoids.

5α-Tetrahydrocorticosterone (5αTHB) is an effective topical anti-inflammatory agent in mouse, with less propensity to cause skin thinning and impede new blood vessel growth compared with corticosterone. Its anti-inflammatory effects were not prevented by RU38486, a glucocorticoid receptor antagonist, suggesting alternative mechanisms. The hypothesis that 5αTHB directly inhibits angiogenesis to a lesser extent than hydrocortisone was tested, focussing on glucocorticoid receptor mediated actions. New vessel growth from aortae from C57BL/6 male mice was monitored in culture, in the presence of 5αTHB, hydrocortisone (mixed glucocorticoid/mineralocorticoid receptor agonist) or the selective glucocorticoid receptor agonist dexamethasone. Transcript profiles were studied, as was the role of the glucocorticoid receptor, using the antagonist, RU38486. Ex vivo, 5αTHB suppressed vessel growth from aortic rings, but was less potent than hydrocortisone (EC50 2512 nM 5αTHB, versus 762 nM hydrocortisone). In contrast to conventional glucocorticoids, 5αTHB did not alter expression of genes related to extracellular matrix integrity or inflammatory signalling, but caused a small increase in Per1 transcript, and decreased transcript abundance of Pecam1 gene. RU38486 did not antagonise the residual effects of 5αTHB to suppress vessel growth or regulate gene expression, but modified effects of dexamethasone. 5αTHB did not alter expression of glucocorticoid-regulated genes Fkbp51 and Hsd11b1, unlike hydrocortisone and dexamethasone. In conclusion, compared with hydrocortisone, 5αTHB exhibits limited suppression of angiogenesis, at least directly in blood vessels and probably independent of the glucocorticoid receptor. Discriminating the mechanisms employed by 5αTHB may provide the basis for the development of novel safer anti-inflammatory drugs for topical use.

A Rat Model of Post-Traumatic Stress Syndrome Causes Phenotype-Associated Morphological Changes and Hypofunction of the Adrenal Gland.

BACKGROUND: Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production. METHODS: Rats were exposed to PSS for ten days. Thirty days later, the rats' anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex. RESULTS: Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxiety phenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats. CONCLUSION: Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder.

Modulation of 11ß-hydroxysteroid dehydrogenase functions by the cloud of endogenous metabolites in a local microenvironment: The glycyrrhetinic acid-like factor (GALF) hypothesis.

11ß-Hydroxysteroid dehydrogenase (11ß-HSD)-dependent conversion of cortisol to cortisone and corticosterone to 11-dehydrocorticosterone are essential in regulating transcriptional activities of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Inhibition of 11ß-HSD by glycyrrhetinic acid metabolites, bioactive components of licorice, causes sodium retention and potassium loss, with hypertension characterized by low renin and aldosterone. Essential hypertension is a major disease, mostly with unknown underlying mechanisms. Here, we discuss a putative mechanism for essential hypertension, the concept that endogenous steroidal compounds acting as glycyrrhetinic acid-like factors (GALFs) inhibit 11ß-HSD dehydrogenase, and allow for glucocorticoid-induced MR and GR activation with resulting hypertension. Initially, several metabolites of adrenally produced glucocorticoids and mineralocorticoids were shown to be potent 11ß-HSD inhibitors. Such GALFs include modifications in the A-ring and/or at positions 3, 7 and 21 of the steroid backbone. These metabolites may be formed in peripheral tissues or by gut microbiota. More recently, metabolites of 11ß-hydroxy-Δ4androstene-3,17-dione and 7-oxygenated oxysterols have been identified as potent 11ß-HSD inhibitors. In a living system, 11ß-HSD isoforms are not exposed to a single substrate but to several substrates, cofactors, and various inhibitors simultaneously, all at different concentrations depending on physical state, tissue and cell type. We propose that this "cloud" of steroids and steroid-like substances in the microenvironment determines the 11ß-HSD-dependent control of MR and GR activity. A dysregulated composition of this cloud of metabolites in the respective microenvironment needs to be taken into account when investigating disease mechanisms, for forms of low renin, low aldosterone hypertension.

Ancient fishes and the functional evolution of the corticosteroid stress response in vertebrates.

The neuroendocrine mechanism underlying stress responses in vertebrates is hypothesized to be highly conserved and evolutionarily ancient. Indeed, elements of this mechanism, from the brain to steroidogenic tissue, are present in all vertebrate groups; yet, evidence of the function and even identity of some elements of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is equivocal among the most basal vertebrates. The purpose of this review is to discuss the functional evolution of the HPA/I axis in vertebrates with a focus on our understanding of this neuroendocrine mechanism in the most ancient vertebrates: the agnathan (i.e., hagfish and lamprey) and chondrichthyan fishes (i.e., sharks, rays, and chimeras). A review of the current literature presents evidence of a conserved HPA/I axis in jawed vertebrates (i.e., gnathostomes); yet, available data in jawless (i.e., agnathan) and chondrichthyan fishes are limited. Neuroendocrine regulation of corticosteroidogenesis in agnathans and chondrichthyans appears to function through similar pathways as in bony fishes and tetrapods; however, key elements have yet to be identified and the involvement of melanotropins and gonadotropin-releasing hormone in the stress axis in these ancient fishes warrants further investigation. Further, the identities of physiological glucocorticoids are uncertain in hagfishes, chondrichthyans, and even coelacanths. Resolving these and other knowledge gaps in the stress response of ancient fishes will be significant for advancing knowledge of the evolutionary origins of the vertebrate stress response.

Carbonyl reductase 1 amplifies glucocorticoid action in adipose tissue and impairs glucose tolerance in lean mice.

OBJECTIVE: Carbonyl reductase 1 (Cbr1), a recently discovered contributor to tissue glucocorticoid metabolism converting corticosterone to 20ß-dihydrocorticosterone (20ß-DHB), is upregulated in adipose tissue of obese humans and mice and may contribute to cardiometabolic complications of obesity. This study tested the hypothesis that Cbr1-mediated glucocorticoid metabolism influences glucocorticoid and mineralocorticoid receptor activation in adipose tissue and impacts glucose homeostasis in lean and obese states. METHODS: The actions of 20ß-DHB on corticosteroid receptors in adipose tissue were investigated first using a combination of in silico, in vitro, and transcriptomic techniques and then in vivo administration in combination with receptor antagonists. Mice lacking one Cbr1 allele and mice overexpressing Cbr1 in their adipose tissue underwent metabolic phenotyping before and after induction of obesity with high-fat feeding. RESULTS: 20ß-DHB activated both the glucocorticoid and mineralocorticoid receptor in adipose tissue and systemic administration to wild-type mice induced glucose intolerance, an effect that was ameliorated by both glucocorticoid and mineralocorticoid receptor antagonism. Cbr1 haploinsufficient lean male mice had lower fasting glucose and improved glucose tolerance compared with littermate controls, a difference that was abolished by administration of 20ß-DHB and absent in female mice with higher baseline adipose 20ß-DHB concentrations than male mice. Conversely, overexpression of Cbr1 in adipose tissue resulted in worsened glucose tolerance and higher fasting glucose in lean male and female mice. However, neither Cbr1 haploinsfficiency nor adipose overexpression affected glucose dyshomeostasis induced by high-fat feeding. CONCLUSIONS: Carbonyl reductase 1 is a novel regulator of glucocorticoid and mineralocorticoid receptor activation in adipose tissue that influences glucose homeostasis in lean mice.

Measurement of 1α hydroxycorticosterone in the Japanese banded houndshark, Triakis scyllium, following exposure to a series of stressors.

An endocrine glucocorticoid response following exposure to a stressor has been well described for many vertebrates. However, despite demonstration of secondary stress responses in a number of elasmobranchs, our understanding of the endocrine control of these responses is lacking. This is largely due to the unusual structure of the dominant corticosteroid in elasmobranch fish, 1α-hydroxycorticosterone (1α-OH-B). Here we describe plasma extraction and HPLC separation procedures that allowed for the measurement of 1α-OH-B and corticosterone from plasma samples in the cannulated, conscious free-swimming Japanese banded houndshark, Triakis scyllium. While patterns of concentration in the plasma for 1α-OH-B and corticosterone were found to be similar in all experiments conducted, circulating levels of 1α-OH-B were consistently 100-fold greater than circulating levels of corticosterone. Immediately following cannulation surgery, circulating levels of 1α-OH-B increased 7-fold compared to pre-surgery levels, while the levels were 11-fold higher than pre-stress levels 30 min post a repeated handling/air-exposure stress. A three week period of fasting resulted in a 22-fold increase in circulating levels of 1α-OH-B in the banded houndshark. This is the first report of direct measurement of changes in circulating levels of the primary corticosteroid in elasmobranch fish, 1α-OH-B, following exposure to a stressor such as handling/air-exposure. Data indicate the steroid may respond similarly to the classic glucocorticoid response, such as cortisol in teleosts.

Quantitative analysis of 11-dehydrocorticosterone and corticosterone for preclinical studies by liquid chromatography/triple quadrupole mass spectrometry.

RATIONALE: The activity of the glucocorticoid activating enzyme 11ß-hydroxysteroid dehydrogenase type-1 (11ßHSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11ßHSD1 converts inert 11-dehydrocorticosterone (11-DHC) into the active form, corticosterone (CORT). A sensitive, specific liquid chromatography/tandem mass spectrometry method was sought to simultaneously quantify total 11-DHC and total and free CORT in murine plasma for simple assessment of 11ßHSD1 activity in murine models. METHODS: Mass spectrometry parameters were optimised and a method for the chromatographic separation of CORT and 11-DHC was developed. Murine plasma was prepared by 10:1 chloroform liquid-liquid extraction (LLE) for analysis. Limits of quantitation (LOQs), linearity and other method criteria were assessed, according to bioanalytical method validation guidelines. RESULTS: Reliable separation of 11-DHC and CORT was achieved using an ACE Excel 2 C18-AR (2.1 × 150 mm; 2 µm) fused core column at 25°C, with an acidified water/acetonitrile gradient over 10 min. Analytes were detected by multiple reaction monitoring after positive electrospray ionisation (m/z 345.1.1 ➔ 121.2, m/z 347.1 ➔ 121.1 for 11-DHC and CORT, respectively). The LOQs were 0.25 and 0.20 ng/mL for 11-DHC and CORT, respectively. CONCLUSIONS: This LC/MS method is suitable for the reliable analysis of 11-DHC and CORT following simple LLE of murine plasma, bringing preclinical analysis in line with recommendations for clinical endocrinology and biochemistry.

Novel Endogenous Ligands of Aryl Hydrocarbon Receptor Mediate Neural Development and Differentiation of Neuroblastoma.

Aryl hydrocarbon receptor (AHR) signaling has been suggested to play roles in various physiological functions independent of its xenobiotic activity, including cell cycle regulation, immune response, and embryonic development. Several endogenous ligands were also identified by high-throughput screening techniques. However, the mechanism by which these molecules mediate AHR signaling in certain functions is still elusive. In this study, we investigated the possible pathway through which AHR and its endogenous ligands regulate neural development. We first identified two neuroactive steroids, 3α,5α-tetrahydrocorticosterone and 3α,5ß-tetrahydrocorticosterone (5α- and 5ß-THB), as novel AHR endogenous ligands through the use of an ultrasensitive dioxin-like compound bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). We then treated zebrafish embryos with 5α- and 5ß-THB, which enhance the expression of neurogenesis marker HuC. Furthermore, 5α- and 5ß-THB both enhanced the expression of myelinating glial cell markers, sex determining region Y-box 10 (Sox10), and myelin-associated proteins myelin basic protein (Mbp) and improved the mobility of zebrafish larvae via the Ahr2 pathway. These results indicated that AHR mediates zebrafish neurogenesis and gliogenesis, especially the differentiation of oligodendrocyte or Schwann cells. Additionally, we showed that these molecules may induce neuroblastoma (NB) cell differentiation suggesting therapeutic potential of 5α- and 5ß-THB in NB treatment. In summary, our results reveal that 5α- and 5ß-THB are endogenous ligands of AHR and have therapeutic potential for NB treatment. By the interaction with THB, AHR signaling regulates various aspects of neural development.

Measurement of 11-dehydrocorticosterone in mice, rats and songbirds: Effects of age, sex and stress.

Glucocorticoids (GCs) are secreted into the blood by the adrenal glands and are also locally-produced by organs such as the lymphoid organs (bone marrow, thymus, and spleen). Corticosterone is the primary circulating GC in many species, including mice, rats and birds. Within lymphoid organs, corticosterone can be locally produced from the inactive metabolite, 11-dehydrocorticosterone (DHC). However, very little is known about endogenous DHC levels, and no immunoassays are currently available to measure DHC. Here, we developed an easy-to-use and inexpensive immunoassay to measure DHC that is accurate, precise, sensitive, and specific. The DHC immunoassay was validated in multiple ways, including comparison with a mass spectrometry assay. After assay validations, we demonstrated the usefulness of this immunoassay by measuring DHC (and corticosterone) in mice, rats and song sparrows. Overall, corticosterone levels were higher than DHC levels across species. In Study 1, using mice, we measured steroids in whole blood and lymphoid organs at postnatal day (PND) 5, PND23, and PND90. Corticosterone and DHC showed distinct tissue-specific patterns across development. In Studies 2 and 3, we measured circulating corticosterone and DHC in adult rats and song sparrows, before and after restraint stress. In rats and song sparrows, restraint stress rapidly increased circulating levels of both steroids. This novel DHC immunoassay revealed major changes in DHC concentrations during development and in response to stress, which have important implications for understanding GC physiology, effects of stress on immune function, and regulation of local GC levels.

Influence of corticosterone on growth, home-cage activity, wheel running, and aerobic capacity in house mice selectively bred for high voluntary wheel-running behavior.

Glucocorticoids, a class of metabolic hormones, impact a wide range of traits (e.g., behavior, skeletal growth, muscle maintenance, glucose metabolism), and variation in concentrations of circulating glucocorticoids (such as corticosterone), at the level of natural individual variation, in relation to endocrine disorders, or from exogenous supplementation, have manifold effects. Changes in circulating corticosterone concentrations can also impact multiple aspects of locomotor behavior, including both motivation and physical ability for exercise. To examine further the role of corticosterone in locomotor behavior and associated traits, we utilized laboratory house mice from a long-term experiment that selectively breeds for high levels of voluntary exercise. As compared with four non-selected control (C) lines, mice from the four replicate High Runner (HR) lines have ~2-fold higher baseline circulating corticosterone concentrations as well as ~3-fold higher voluntary wheel running on a daily basis, higher home-cage activity when deprived of wheels, higher maximal aerobic capacity, and smaller body size; potentially, all of these differences could be modulated by circulating corticosterone. We administered 50 µg/mL corticosterone-21-hemisuccinate in the drinking water of both HR and C male mice from weaning through ~8 weeks of age. As compared with mice from C lines, HR mice had higher endogenous corticosterone levels; higher daily wheel-running distance, duration, and speed; higher maximal oxygen consumption during forced exercise (VO2max); spent more time in the closed arms of an elevated plus maze; and had larger reproductive fat pads. For both HR and C mice, corticosterone treatment strongly suppressed endogenous circulating corticosterone levels, decreased growth rate and adult body mass, increased food and water consumption (both adjusted for body mass), increased entries into closed arms of an elevated plus maze, decreased home-cage activity (total and average intensity), decreased wheel-running distance and maximum speed, and decreased VO2max. At the suborganismal level, corticosterone treatment decreased relative adrenal, liver, and triceps surae muscle mass, as well as tail length, but increased both subdermal and reproductive fat pad masses, as well as hematocrit. Overall, the responses of both HR and C mice to corticosterone supplementation were "negative" from a health perspective. These results have significant implications for understanding both the evolution of baseline corticosterone levels and stress-related effects on activity levels. They also suggest that patients experiencing extended periods of glucocorticoid treatment might benefit from attempts to increase their physical activity as an adjuvant.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

Glucocorticoids Reprogram ß-Cell Signaling to Preserve Insulin Secretion.

Excessive glucocorticoid exposure has been shown to be deleterious for pancreatic ß-cell function and insulin release. However, glucocorticoids at physiological levels are essential for many homeostatic processes, including glycemic control. We show that corticosterone and cortisol and their less active precursors 11-dehydrocorticosterone (11-DHC) and cortisone suppress voltage-dependent Ca2+ channel function and Ca2+ fluxes in rodent as well as in human ß-cells. However, insulin secretion, maximal ATP/ADP responses to glucose, and ß-cell identity were all unaffected. Further examination revealed the upregulation of parallel amplifying cAMP signals and an increase in the number of membrane-docked insulin secretory granules. Effects of 11-DHC could be prevented by lipotoxicity and were associated with paracrine regulation of glucocorticoid activity because global deletion of 11ß-hydroxysteroid dehydrogenase type 1 normalized Ca2+ and cAMP responses. Thus, we have identified an enzymatically amplified feedback loop whereby glucocorticoids boost cAMP to maintain insulin secretion in the face of perturbed ionic signals. Failure of this protective mechanism may contribute to diabetes in states of glucocorticoid excess, such as Cushing syndrome, which are associated with frank dyslipidemia.

Biosynthesis of 1α-hydroxycorticosterone in the winter skate Leucoraja ocellata: evidence to suggest a novel steroidogenic route.

The present study explores the ability of intracellular bacteria within the renal-inter-renal tissue of the winter skate Leucoraja ocellata to metabolize steroids and contribute to the synthesis of the novel elasmobranch corticosteroid, 1α-hydroxycorticosterone (1α-OH-B). Despite the rarity of C1 hydroxylation noted in the original identification of 1α-OH-B, literature provides evidence for steroid C1 hydroxylation by micro-organisms. Eight ureolytic bacterial isolates were identified in the renal-inter-renal tissue of L. ocellata, the latter being the site of 1α-OH-B synthesis. From incubations of bacterial isolates with known amounts of potential 1α-OH-B precursors, one isolate UM008 of the genus Rhodococcus was seen to metabolize corticosteroids and produce novel products via HPLC analysis. Cations Zn2+ and Fe3+ altered metabolism of certain steroid precursors, suggesting inhibition of Rhodococcus steroid catabolism. Genome sequencing of UM008 identified strong sequence and structural homology to that of Rhodococcus erythropolis PR4. A complete enzymatic pathway for steroid-ring oxidation as documented within other Actinobacteria was identified within the UM008 genome. This study highlights the potential role of Rhodococcus bacteria in steroid metabolism and proposes a novel alternative pathway for 1α-OH-B synthesis, suggesting a unique form of mutualism between intracellular bacteria and their elasmobranch host.

11ß-HSD1 suppresses cardiac fibroblast CXCL2, CXCL5 and neutrophil recruitment to the heart post MI.

We have previously demonstrated that neutrophil recruitment to the heart following myocardial infarction (MI) is enhanced in mice lacking 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) that regenerates active glucocorticoid within cells from intrinsically inert metabolites. The present study aimed to identify the mechanism of regulation. In a mouse model of MI, neutrophil mobilization to blood and recruitment to the heart were higher in 11ß-HSD1-deficient (Hsd11b1-/- ) relative to wild-type (WT) mice, despite similar initial injury and circulating glucocorticoid. In bone marrow chimeric mice, neutrophil mobilization was increased when 11ß-HSD1 was absent from host cells, but not when absent from donor bone marrow-derived cells. Consistent with a role for 11ß-HSD1 in 'host' myocardium, gene expression of a subset of neutrophil chemoattractants, including the chemokines Cxcl2 and Cxcl5, was selectively increased in the myocardium of Hsd11b1-/- mice relative to WT. SM22α-Cre directed disruption of Hsd11b1 in smooth muscle and cardiomyocytes had no effect on neutrophil recruitment. Expression of Cxcl2 and Cxcl5 was elevated in fibroblast fractions isolated from hearts of Hsd11b1-/- mice post MI and provision of either corticosterone or of the 11ß-HSD1 substrate, 11-dehydrocorticosterone, to cultured murine cardiac fibroblasts suppressed IL-1α-induced expression of Cxcl2 and Cxcl5 These data identify suppression of CXCL2 and CXCL5 chemoattractant expression by 11ß-HSD1 as a novel mechanism with potential for regulation of neutrophil recruitment to the injured myocardium, and cardiac fibroblasts as a key site for intracellular glucocorticoid regeneration during acute inflammation following myocardial injury.

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 24h, although at a five fold higher dose, but in contrast did not have any effects after 6h. Pre-treatment with the glucocorticoid receptor antagonist RU486 attenuated the effect of corticosterone on swelling at 24h, but not that of 5αTHB. After 24h 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.

Different Efficiency of Liposomal Forms with Hydrophilic and Hydrophobic Antitumor Agents in Relation to Solid Transplants of Mouse Tumor and Its Metastases in the Liver.

Experiments were performed on the model of transplanted mouse tumor with high incidence of liver metastases. Hydrophilic drug cycloplatam (injected intravenously in liposomes) was more potent than "free cycloplatam" (injected intravenously or intraperitoneally in physiological saline) in inhibiting the growth of natural and experimental metastases in the liver. By contrast, liposomal cycloplatam had lower efficiency than free cycloplatam in suppressing the growth of solid tumor. Liposomal and free cortifen (hydrophobic hormonal cytostatic) produced nearly the same effects on solid tumor growth. Our results suggest that liposomal forms of hydrophobic compounds producing nonselective effect on tumor cells (e.g., actinomycin D or Cosmegen), should not have advantages over free forms.

Obesity Disrupts the Rhythmic Profiles of Maternal and Fetal Progesterone in Rat Pregnancy.

Maternal obesity increases the risk of abnormal fetal growth, but the underlying mechanisms remain unclear. Because steroid hormones regulate fetal growth, and both pregnancy and obesity markedly alter circadian biology, we hypothesized that maternal obesity disrupts the normal rhythmic profiles of steroid hormones in rat pregnancy. Obesity was established by cafeteria (CAF) feeding for 8 wk prior to mating and throughout pregnancy. Control (CON) animals had ad libitum access to chow. Daily profiles of plasma corticosterone, 11-dehydrocorticosterone, progesterone, and testosterone were measured at Days 15 and 21 of gestation (term = 23 days) in maternal (both days) and fetal (Day 21) plasma. CAF mothers exhibited increased adiposity relative to CON and showed fetal and placental growth restriction. There was no change, however, in total fetal or placental mass due to slightly larger litter sizes in CAF. Nocturnal declines in progesterone were observed in maternal (39% lower) and fetal (45% lower) plasma in CON animals, but these were absent in CAF animals. CAF mothers were hyperlipidemic at both days of gestation, but this effect was isolated to the dark period at Day 21. CAF maternal testosterone was slightly lower at Day 15 (8%) but increased above CON by Day 21 (16%). Despite elevated maternal testosterone, male fetal testosterone was suppressed by obesity on Day 21. Neither maternal nor fetal glucocorticoid profiles were affected by obesity. In conclusion, obesity disrupts rhythmic profiles of maternal and fetal progesterone, preventing the normal nocturnal decline. Obesity subtly changed testosterone profiles but did not alter maternal and fetal glucocorticoids.

Local cortisol/corticosterone activation in skin physiology and pathology.

Cortisol and corticosterone are the endogenous glucocorticoids (GCs) in humans and rodents, respectively. Systemic GC is released through the hypothalamic-pituitary-adrenal (HPA) axis in response to various stressors. Over the last decade, extra-adrenal production/activation of cortisol/corticosterone has been reported in many tissues. The enzyme that catalyzes the conversion of hormonally inactive cortisone/11-dehydrocorticosterone (11-DHC) into active cortisol/corticosterone in cells is 11ß-hydroxysteroid dehydrogenase (11ß-HSD). The 11ß-HSD1 isoform is predominantly a reductase, which catalyzes nicotinamide adenine dinucleotide phosphate hydrogen-dependent conversion of cortisone/11-DHC to cortisol/corticosterone, and is widely expressed and present at the highest levels in the liver, lungs, adipose tissues, ovaries, and central nervous system. The 11ß-HSD2 isoform, which catalyzes nicotinamide adenine dinucleotide+-dependent inactivation of cortisol/corticosterone to cortisone/11-DHC, is highly expressed in distal nephrons, the colon, sweat glands, and the placenta. In healthy skin, 11ß-HSD1 is expressed in the epidermis and in dermal fibroblasts. On the other hand, 11ß-HSD2 is expressed in sweat glands but not in the epidermis. The role of 11ß-HSD in skin physiology and pathology has been reported recently. In this review, we summarize the recently reported role of 11ß-HSD in the skin, focusing on its function in cell proliferation, wound healing, inflammation, and aging.

Stress and hypertensive disease: adrenals as a link. Experimental study on hypertensive ISIAH rat strain.

OBJECTIVE: Association between stress and hypertensive disease is still a matter of debate. Can stress be the cause of hypertensive disease and, if so, what mechanisms are involved? To clarify this question, the Inherited stress-induced arterial hypertensive rat strain (ISIAH rat strain) with a stress related arterial hypertension was developed by selection for the enhanced blood pressure response to 0.5 h restraint stress. The main intention of this work is to confirm that the adrenals are a main link between stress and hypertensive disease. METHODS: Hypertensive ISIAH and normotensive WAG rats have been studied. The in vivo secretion rate of corticosterone, aldosterone, 11-Deoxycorticosterone (DOC), and 11-dehydrocorticosterone was measured in anesthetized rats by adrenal vein cannulation. The Dexamethasone/Adrenocorticotropic hormone (DEX/ACTH) test was performed and mRNA expression of Cyp11b1 and Cyp11b2 genes in adrenals was evaluated by real-time PCR. RESULTS: An increased secretion rate of corticosterone and DOC and higher peripheral plasma aldosterone concentration in ISIAH rats were revealed. Response of plasma aldosterone to the surgical stress (adrenal vein cannulation) in the ISIAH rats was significantly higher. The increase of corticosterone and aldosterone in response to ACTH was also higher in hypertensive rats. The basal mRNA expression of both Cyp11b1 and Cyp11b2 genes was increased in the ISIAH rats. The ratio 11-dehydrocorticosterone/corticosterone in ISIAH rats was low which indicates the weakening of 11-beta-Hydroxysteroid dehydrogenase (11-beta-HSD) type 2 converting corticosterone to cortisone. CONCLUSION: ISIAH rats may serve as a living proof that stress may produce sustained hypertension, and genetically determined enhanced stress responsiveness of corticosterone and, especially, aldosterone may play a crucial role in the mechanism of hypertension development.