Thesis (M.S. in biochemistry)--Washington State University, August 2006. / Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115-121). Also available for download via the World Wide Web; free to University of Oregon users.

Glucocorticoids (GC) have a negative effect on age-associated cognitive decline and the GC metabolising enzyme 11β-hydroxysteroid dehydrogenase Type 1 (11β- HSD1) plays a key role in these effects. Increased glucocorticoids exert detrimental effects on the volume and function of brain regions such as the prefrontal cortex (PFC) and hippocampus that are necessary for cognitive functions such as memory and working memory. Previous research has identified changes in cell populations, metabolite levels and structure within the brain as well as altered levels of inflammation with age, and studies have suggested these biomarkers to be associated with cognitive impairments. Aged mice with a deletion in 11ß-HSD1 (11β-HSD1-/- mice), resulting in lower levels of glucocorticoids within the brain, exhibit attenuated cognitive decline in hippocampal dependent spatial learning and memory with age. However, the mechanisms through which 11β-HSD1 contribute to age-associated cognitive decline remain unknown. However, previous genetic models of 11β-HSD1- /- mice have demonstrated residual 11β-HSD1 activity in the brain which may still exert some effects on cognitive processes. Furthermore, the effect of 11ß-HSD1 on working memory – a more cognitively demanding process essential for everyday decision making - has yet to be determined. This thesis tests the hypothesis that glucocorticoid action mediates age-associated cognitive impairment in spatial learning and memory and spatial working memory through alterations in cell activity, brain metabolite levels and neuroinflammatory processes. Therefore, we aimed to investigate if complete lifelong 11β-HSD1 deficiency would protect against age-associated working memory deficits as well as spatial learning and memory deficits, and its effect on associated neural markers. In particular, we determined changes in hippocampal metabolite levels, cell activity and inflammation as a function of ageing in a longitudinal manner. At 6, 12, 18 and 22 months, male 11β- HSD1-/- and C57BL/6J control mice were cognitively assessed in the Morris Water Maze (MWM) and Radial Arm Water Maze (RAWM) – tests for spatial reference memory and spatial working memory respectively. Magnetic resonance spectroscopy (1H-MRS) was performed to examine the hippocampal metabolite profile in the same mice at 6, 18 and 22 months. Following their final scan, mice were culled and brains dissected for analysis. Results revealed unaltered spatial learning with age in C57BL/6J and 11β-HSD1-/- mice and pointed to a development of alternative strategies for task completion as a result of repeated testing. Spatial memory was more susceptible to age-associated effects with impairments in wild-type mice but not 11β-HSD1-/- mice, though not completely immune from the effects of repeated testing. These impairments were correlated with glutamate/glutamine levels and glial fibrillary acidic protein (GFAP), whilst GFAP was further correlated with 11β-HSD1 protein expression. Working memory was impaired with age in both 11β-HSD1-/- and wild-type mice, suggesting 11β-HSD1 deletion may be detrimental to cognitive processes in the prefrontal cortex. In conclusion, impaired memory with age may be attributed to increased glial reactivity and altered glutamate/glutamine cycling in the hippocampus, and lifelong removal of 11β-HSD1 may alter these processes. However, lifelong removal of 11β-HSD1 may not be as beneficial to working memory processes suggesting that 11β-HSD1 and glucocorticoid action play a key role in working memory processes.

Background Sarcopenia and age-related cognitive decline (ARCD) are important age-related conditions which significantly impact upon the quality of life of older adults. ARCD is a well-established research area, whereas sarcopenia is a relatively new field. Research into the inter-relationships between them and possible common underlying mechanistic processes is lacking. Methods Several research techniques were used: a large systematic review; the development of an image analysis technique to measure neck muscle size on volumetric MR brain scans; the subsequent use of the technique in elderly cohort studies; statistical modelling to investigate the role of glucocorticoids in sarcopenia; and an invasive clinical study to develop a novel technique to measure the activity of 11beta-hydroxysteroid dehydrogenase (11βHSD1) in the human brain in vivo. Results I consistently found a relationship between: some measures of brain structure and muscle size; markers of brain structure and muscle function, mostly grip strength and gait speed; and cognition and muscle function. However, I found no relationship between current cognition and muscle size in any of the above studies. Cortisol was identified as a possible explanatory factor in the relationship between both cognition and brain volume with gait speed. I found an association between markers of immunosenescence and sarcopenia (neck muscle CSA and grip strength) and an association between expression of the cortisol amplifying enzyme 11βHSD1 and quadriceps strength. I developed a technique to measure 11βHSD1 activity across the human brain, which found that the amount of cortisol produced within the brain was not detectable and highlighted the asymmetries within the cerebrovascular venous system. Conclusions Further longitudinal studies looking at the association between sarcopenia and ARCD are now required to investigate these important relationships further and hopefully this will lead to improved therapeutic options.

Fetal growth restriction (FGR) signifies that the fetus has not achieved its growth potential and is associated with increased perinatal mortality and morbidity. The exact aetiology of FGR, in the absence of any identifiable fetal and maternal factors, remains unclear and is attributed to placental insufficiency. The FGR placenta has a characteristic phenotype including: increased resistance in the fetoplacental circulation, an alteration in trophoblast cell turnover and reduced activity of placental nutrient transport systems, the best characterised being the amino acid transporter, system A. The placenta strongly expresses the cortisol inactivating enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). 11β-HSD2 activity was reduced in placentas from pregnancies complicated by FGR, suggesting increased exposure of the fetoplacental unit to maternal cortisol. In animal models, excessive exposure to glucocorticoids (GCs) is associated with a reduction in both fetal and placental weight. This reduction in placental weight was associated with abnormalities in placental function, consistent with those observed in the FGR placenta. This PhD investigated whether excess GC exposure during pregnancy is responsible placental insufficiency in human pregnancies and tested the hypotheses that excess GC exposure adversely affects placental vascular tone, trophoblast cell turnover and activity of the amino acid transporter, system A. Term placentas were collected from uncomplicated pregnancies and first trimester placental samples were obtained following elective surgical termination of pregnancy. Wire myography was used to explore the acute and chronic effects of GCs on term chorionic plate artery (CPA) function. The impact of GC treatment on trophoblast cell turnover in both first trimester and term placenta was investigated using the placental explant system. The effect of GCs on the activity of the system A transporter was also investigated in term explants and in isolated cytotrophoblasts where the expression of 11β-HSD2 was reduced using siRNA. Gene microarray studies on first trimester placental explants treated with GCs were utilised to identify genes regulated by GCs. Blunted constriction to thromboxane A2 was observed following acute GC treatment, whilst chronic exposure resulted in enhanced vasoconstriction, mimicking the altered reactivity of CPAs from pregnancies complicated by FGR. GC excess in first trimester placental explants increased apoptosis and decreased proliferation, thereby replicating the disordered turnover of the trophoblast observed in FGR placentas. No demonstrable effect was observed in cell turnover or system A activity in term placental explants treated with GCs, however, these experiments were hindered by the in-vitro regeneration of the syncytiotrophoblast in the model employed. The attenuation of 11β-HSD2 activity observed in FGR placentas was replicated in term primary cytotrophoblasts utilising siRNA to knock-down expression of 11β-HSD2. Preliminary results suggested an increase in system A activity in response to cortisol. Gene microarray studies identified a significant number of genes (~500) that were regulated by dexamethasone, confirming that GCs have an impact on many aspects of placental function. Potential mediators for the characteristic features of the FGR placenta replicated here in response to GC treatment were identified and validated at the mRNA level. The studies described in this thesis support the hypotheses that GC excess within the placenta contributes to the development of raised vascular resistance in the fetoplacental circulation and the disordered trophoblast turnover in placentas from pregnancies complicated by FGR. However, with the preliminary studies performed, the hypothesis that elevated levels of GCs contribute to the reduced placental amino acid transfer by the system A transporter in the FGR placenta can not be confidently disproven.

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Human endothelium is capable of expressing a variety of molecules, including cytokines and growth factors, critical to inflammation. This aspect of coronary endothelium has not been studied in detail. In this study, we report, for the first time, expression of multifunctional cytokines by human coronary artery endothelial cells (HCAEC) and their regulation by inflammatory cytokines and glucocorticoids. We also compared expression of cytokine transcripts in two additional cell lines derived from pulmonary artery (HPAEC) and umbilical vein (HUVEC) endothelium. HCAEC expressed transcripts for interleukin 5 (IL-5), IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) constitutively. Induction of IL-1α, IL-6, granulocyte- macrophage colony-stimulating factor (GM-CSF), and MCP-1 was seen following treatment with TNFα. We found no expression of IL-1RA, IL-2, IL-4, IL-13, TNF-α, or IFN-γ in HCAEC. IL-1β and TNF-α synergistically induced IL-6 and GM-CSF and additively induced IL-8 and MCP-1 production, while IL-2, IL- 10, IFN-α, and IFN-γ had little or no additional effects. Interestingly, no IL-1α or IL-5 protein product was found even after maximal stimulation of HCAEC. No significant differences were seen in the profile of cytokine genes expressed by HCAEC, HPAEC, or HUVEC. Glucocorticoids inhibited IL-8 production from all three cell lines. This study demonstrates that human coronary endothelial cells are capable of expressing a wide variety of multifunctional cytokines which may be of relevance to vascular inflammation.

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