COPD is a condition characterised by chronic inflammation in which macrophages arethought to play a central role. Corticosteroids are the most widely used antiinflammatoryagents to treat COPD. There is a subset of inflammatory mediatorswhich are corticosteroid insensitive and so there is a need for novel anti-inflammatorytherapies to treat COPD. One such target is the liver X receptor (LXR), a cholesterolsensing nuclear hormone receptor with anti-inflammatory properties. Before investigating the anti-inflammatory potential of LXR, I aimed to validate thealveolar macrophage in vitro culture model. I investigated the effect of differentculture times on unstimulated and stimulated cytokine release, dexamethasoneinhibition of cytokine release, and transcription factor phosphorylation in alveolarmacrophages from COPD patients and controls. I found that freshly isolatedmacrophages release higher levels of cytokines, are less responsive to dexamethasoneand have increased levels of phosphorylated p38 MAPK.I next investigated LXR gene and protein expression levels in alveolar macrophages andwhole lung tissue from COPD patients and controls, the effect of LXR activation on thesuppression of inflammatory mediators from LPS stimulated COPD alveolarmacrophages, and the effect of LXR activation on the induction of genes associatedwith alternative macrophage polarisation. The levels of LXR mRNA were significantlyincreased in whole lung tissue extracts in COPD patients and smokers compared tonever smokers. The expression of LXR protein was significantly increased in smallairway epithelium and alveolar epithelium in COPD patients compared to controls. Nodifferences in LXR mRNA and protein levels were observed in alveolar macrophagesbetween patient groups. The LXR agonist GW3965 significantly induced the expressionof the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. InLPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10and CCL5, whilst stimulating IL-10 production. GW3965 did not significantly suppressthe production of TNFα, IL-1β, or CXCL8. The major finding is that LXR activation hasanti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolarmacrophages. Finally, I investigated whether dysfunctional lipid homeostasis is a feature of COPDalveolar macrophages. I found that alveolar macrophages from current smokers withand without COPD had increased levels of neutral lipids compared to never smokersand ex-smokers with and without COPD. Dysfunctional lipid homeostasis may play arole in COPD.

Chez les mammiferes, la lipogenese ou synthese de novo des acides gras joue un rôle essentiel a l'homeostasie energetique. Elle est particulierement active dans le foie. Le Liver X Receptor (LXR) est un recepteur nucleaire de classe II qui est implique dans la regulation de l'expression de genes importants dans cette voie metabolique. Au niveau hepatique, LXR regule directement l'expression de certains genes de la lipogenese et aussi l'expression des facteurs de transcription SREBP-1c et ChREBP intervenant respectivement dans la reponse hepatique a l'insuline et au glucose. Les ligands naturels de LXR sont les oxysterols, des derives oxygenes du cholesterol. Aussi, LXR est avant tout considere et connu comme un senseur du cholesterol. Au cours de ces travaux nous nous sommes interesses in vivo au role de LXR dans la regulation transcriptionnelle de la lipogenese hepatique en fonction de differents stimuli: pharmacologiques, inflammatoires et nutritionnels. Par une approche pharmacologique, nous avons etudie la regulation croisee avec entre LXR et le recepteur active par les proliferateurs de peroxisome (PPAR . Nous avons aussi montre que l'inflammation intestinale est un puissant inhibiteur de la lipogenese hepatique. Enfin, nous avons mis en evidence le role de LXR dans la regulation de la lipogenese en reponse a une carence en acides gras essentiels et a un regime riche en fructose. / In mammals, lipogenesis or de novo fatty acid synthesis plays an essential part in energy homeostasis. It is particularly active in the liver. The Liver X Receptor (LXR) is a class II nuclear receptor that regulates the expression of important genes involved in this pathway. In the liver, LXR directly controls the expression of lipogenic genes and also the expression of transcription factors such as SREBP-1c and ChREBP required for the hepatic response to insulin and glucose respectively. Natural ligands for LXR are oxysterols, which are oxygenated derivatives of cholesterol. Therefore, LXR is primarily considered and known as a cholesterol sensor. In this work, we were interested in the role of LXR in the transcriptional control of hepatic lipogenesis in vivo in response to distinct stimuli: pharmacological agonists, gut inflammation and changes in diet composition. Through a pharmacological study, we highlighted the cross-talk between LXR signaling and the regulation of the Peroxisome Proliferator Activated Receptor (PPAR ). We have also evidenced that experimentally induced colitis induces a potent inhibition of hepatic lipogenesis. Finally, we have shown that LXR is involved in the regulation of lipogenesis in response to essential fatty acid deficiency and to high fructose.

The role cholesterol plays in the brain has long been underappreciated even though the brain contains a disproportionately high percentage of body cholesterol. Recent studies have found a link between the dysregulation of lipid metabolism and the risk of acquiring Alzheimer’s disease (AD) as well as a predisposition to cognitive decline. The goal of these studies was to elucidate the possible role lipid metabolism plays in pathological and normal brain aging by pharmacologically manipulating lipid metabolism and determining effects on key hippocampal biomarkers of AD and age-related cognitive decline. One series of experiments used an agonist (TO901317) to the liver X receptor (LXR) in two transgenic AD mouse models. Chronic LXR activation reduced AD associated pathology and improved cognitive performance in AD mouse models. However, long-term potentiation (LTP) was not enhanced and peripheral side effects were observed. In another series of experiments the effects of chronically inhibiting cholesterol synthesis on cognitive aging in rats was determined. Animals were treated with either of two commonly prescribed statins, simvastatin or atorvastatin. Simvastatin, the more lipophilic statin, increased LTP and reduced the duration of the afterhyperpolarization (AHP). In addition, simvastatin upregulated key genes of the cholesterol synthesis pathway in the hippocampus as revealed by microarray analyses, but was associated with impaired performance in the Morris Water Maze, a hippocampal dependent task. Atorvastatin, a less lipophilic statin, reduced the AHP, but did not affect LTP or cognitive performance. Atorvastatin modulated a very different set of genes and reduced brain cholesterol more than simvastatin. These results suggest that manipulation of cholesterol metabolism selectively modulates key aspects of AD and brain aging.

The nuclear hormone receptor (NHR) liver X receptor β (LXRβ) has been highlighted as a possible candidate for involvement in ageing by several recent findings. LXRβ is the closest human homologue to the longevity-associated gene daf-12 in the nematode worm and LXRβ haplotypes have been associated with longevity at old ages in a longitudinal human genetic study. Whilst LXRβ is primarily responsible for mediating the effects of LXR oxysterol ligands throughout most of the body, LXRβ is the primary mediator of these effects uniquely in the skin. In this thesis studies are presented on the expression of LXRβ mRNA and protein in human skin, comparing young vs intrinsically (chronologically) aged skin, photoprotected vs photoaged (dueto ultraviolet radiation exposure) skin and untreated vs retinoid-treated photoaged skin, retinoid treatment being a primary clinical intervention for photoageing. In situ hybridisation and quantitative polymerase chain reaction (qPCR) were used to identify LXRβ mRNA and immunofluorescence was used to identify LXRβ protein. These comparisons revealed that both the mRNA and protein expression of LXRβ are highly stable throughout the ageing, photoageing and retinoid treatment of human skin. Previous authors have identified overlap between microarray gene expression datasets in the LXRβ-/- mouse and in normal human skin ageing. In these studies comparisons of different microarray datasets have been conducted with the finding that LXR agonist treatment of mice produces gene regulation patterns with significant overlap to that seen in both ageing and calorie restriction in mice (binomial test; p<0.001). Furthermore, when considering the genes commonly regulated in LXR agonist treatment and ageing, 73% of these genes are regulated in opposite directions. Conversely, when considering genes commonly regulated in LXR agonist treatment and calorie restriction, 70% of these genes are regulated in the same direction. These findings suggest that LXR agonists have possible benefit as ageing therapies, perhaps due to stimulating a calorie restriction-like response. Further work would be necessary to confirm these properties of LXR agonists and to define the roles of LXRβ in the ageing and normal function of human skin.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23814号 / 医科博第135号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 松村 由美, 教授 森本 尚樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Despite advances in healthcare, cardiovascular disease (CVD) remains the leading cause of death in the United States. Elevated levels of plasma cholesterol are highly predictive of CVD and stroke and are the principal driver of atherosclerosis. Unfortunately, current cholesterol lowering agents, such as statins, are not known to reverse atherosclerotic disease once it has been established. In preclinical models, agonists of nuclear receptor, LXR, have been shown to reduce and reverse atherosclerosis. Phytosterols are bioactive non-cholesterol sterols that act as LXR agonists and regulate cholesterol metabolism and transport. We hypothesize that stigmasterol would act as an LXR agonist and alter intestinal cholesterol secretion to promote cholesterol elimination. Mice were fed a control diet, or a diet supplemented with stigmasterol (0.3% w/w) or T0901317 (0.015% w/w), a known LXR agonist. In this experiment we analyzed the sterol content of bile, intestinal perfusate, plasma, and feces. Additionally, the liver and small intestine were analyzed for relative levels of transcripts known to be regulated by LXR. We observed that T0901317 robustly promoted cholesterol elimination and acted as a strong LXR agonist. Stigmasterol also promoted cholesterol elimination but did not alter LXR-dependent gene expression. Stigmasterol promoted transintestinal cholesterol secretion through an LXR-independent pathway.

L’épididyme est un organe accolé au testicule, formé d’un long tubule dont l’épithélium est constitué de 6 types cellulaires différents aux fonctions partiellement connues. Le tubule épididymaire est entouré de muscles lisses participant, par leurs contractions, à l’avancée des spermatozoïdes. Au centre du canal se retrouve le fluide épididymaire dont la composition est régulée par les absorptions et sécrétions de l’épithélium épididymaire. Les gamètes arrivant du testicule sont inaptes à assurer la fécondation. La maturation post-testiculaire des spermatozoïdes, durant leur descente dans le tubule épididymaire, va leur permettre d’acquérir la mobilité et la capacité à reconnaître et féconder un ovule (qualités regroupées sous le terme de “pouvoir fécondant”). Cette maturation se fait grâce aux interactions permanentes des spermatozoïdes avec le fluide. Les LXRs sont des régulateurs majeurs de l’homéostasie du cholestérol, un élément fondamental dans la physiologie de la reproduction puisqu’il est le précurseur des hormones stéroïdes (androgènes) et un régulateur du pouvoir fécondant des spermatozoïdes. Les souris mâles invalidées pour les 2 isoformes de ces récepteurs (lxrα;β-/-) ont une fertilité perturbée en association avec des altérations testiculaires et épididymaires. Ces mâles présentent une infertilité dès 6 mois conduisant à une stérilité complète à 9 mois. Le but de ce travail est de comprendre la relation entre la régulation de l’homéostasie du cholestérol et le phénotype épididymaire de ces souris. A l’âge de 4 mois, alors que les souris mâles lxrα;β-/- sont fertiles, le phénotype est caractérisé par une accumulation de lipides dans certains types cellulaires de l’épithélium et dans les muscles lisses entourant le tubule. Chez les mâles lxrα;β-/- âgés de 9 mois, lorsque le phénotype d’infertilité complète est installé, on observe une destructuration de la partie proximale de l’épididyme comprenant une diminution de la hauteur de l’épithélium, une perte des muscles lisses péritubulaires et la présence de spermatozoïdes non mobiles et malformés dans la lumière du tubule. Mes travaux ont permis de mettre en évidence différents phénomènes : 1/ Une nouvelle fonction d’un type cellulaire particulier, les cellules “apicales”, dans la régulation de l’homéostasie du cholestérol épididymaire. En effet, ces cellules accumulent du cholestérol en association avec la perte de “ABCA1” (ATP Binding Cassette A1), codée par un gène cible de LXR, impliquée dans l’export de l’excédent de cholestérol cellulaire. Suite à cela, ces cellules apicales entrent en apoptose et contribuent à la perte de fonction de l’épithélium. 2/ Une altération des cellules musculaires lisses péritubulaires associée au vieillissement des souris lxrα;β-/-. Ces cellules accumulent elles aussi du cholestérol et se transforment en cellules invasives, les cellules “spumeuses” migrant à travers l’épithélium. Ce phénomène est très proche des phases de développement de la pathologie artérielle liée au cholestérol et au vieillissement, l’athérosclérose. La perte des muscles lisses va limiter la progression des spermatozoïdes et accompagner la perte de fonction des cellules épithéliales qui ne seront plus aptes à effectuer des échanges avec le fluide, empêchant ainsi l’acquisition du pouvoir fécondant des spermatozoïdes. (...) / The epididymis is a long tubule which epithelium is made of 6 different cellular types with partially unknown functions. The epididymal tubule is surrounded by smooth muscles participating, by their contractions, to the sperm progression. At the center of the tubule is present the epididymal fluid which composition is regulated by the absorptions and secretions of the epithelium. The spermatozoa coming from the testis are unable to fertilize an oocyte. Post-testicular maturation of spermatozoa, during their transit through the epididymis, allows them to become motile, to recognize and to fertilize an oocyte. This maturation is due to the constant interaction of the spermatozoon with the epididymal fluid. LXR are the major sensors of cholesterol homeostasis, a fundamental element in reproductive physiology because it is the steroid hormones precursor and a regulator of the sperm fertilizing capacities. LXR deficient mice (lxrα;β-/-) have fertility disorder associated with testicular and epididymal alterations. These males present an infertility since 6 months of age leading to a complete infertility at 9 months of age. The purpose of this work is to understand the relation between cholesterol homeostasis regulation and epididymal sperm maturation. At 4 months of age, the lxrα;β-/- males are fertile but present an epididymal phenotype characterized by neutral lipid accumulations in some epithelial cell types and in the smooth muscle cells surrounding the tubule. At 9 months of age, the totally infertile lxrα;β-/- male, present a complete destructuration of the proximal epididymis associated with a complete shrinking of the epithelium height, the loss of the peritubular smooth muscle cells and the presence of immotile and abnormal spermatozoa in the cauda epididymis. My works put in light : 1/ A novel function of a particular cell type, the “apical cells”, in the regulation of the epididymal cholesterol homeostasis. These cells accumulate cholesteryl esters in association with the loss of the ATP Binding Cassette A1 “ABCA1”, a transporter implicated in the cholesterol efflux. Following this, apical cells become apoptotic participating to the epithelial function loss. 2/ An alteration of peritubular smooth muscle cells associated with the mice aging. These cells also accumulate cholesteryl esters and transdifferentiate into invasive foam cells migrating through the epithelium. This phenomenon is very similar to what is observed in the arterial pathology related to cholesterol and aging, the atherosclerosis. The loss of smooth muscle cells limit the progression of the spermatozoa in the lumen and lead to the loss of function of the epithelial cells which become unable to make any exchange with the fluid, hence disturbing spermatozoa maturation. 3/ A deleterious effect of a cholesterol enriched diet on the fertility in 3-month-old lxrα;β-/- male, which are normally fertile and without epididymal phenotype. When submitted to the diet during 4 weeks, these mice become completely infertile, showing the epididymal phenotype of the 9-month-old lxrα;β-/- male mice. The infertility of these mice is characterized by defects in spermatozoa motility, viability and functional parameters. Our results show, on the one hand, the importance of LXR in the post testicular maturation of spermatozoa and in the maintenance of the integrity and functionality of the epididymis. On the other hand, the data that we collected put in evidence the impact of the alimentation and in particular cholesterol enriched diet on the post-testicular maturation.

Thesis (M.D.) -- University of Texas Southwestern Medical Center at Dallas, 2007. / Vita. Bibliography: pp.28-30

<p>Stroke is the major cause of serious long-term disability with a sufficient acute treatment for only a very limited number of patients. Limited recovery of neurological functions occurs and can be elevated by a permissive post-stroke milieu. Housing animals in an enriched environment modulates regenerative mechanisms in the nonischemic peri-infarct area which might be an attractive target for pharmacological treatments to promote recovery.</p><p>Upon ischemia, cellular lipids are released due to massive cell damage and free lipids significantly contribute to the progression of acute and delayed cell death. The aim of this study was to evalute the effect of enriched environment on lipid metabolism. In particular we characterize the activation of the transcription factor liver X receptor (LXR) in glial scar formation and regulation of cholesterol balance of relevance for functional recovery following stroke.                                      Brain tissues from animals subjected to permanent occlusion of middle cerebral artery (pMCAo) were analysed for LXRα and β protein expression. We found an upregulation and an increased transcriptional activity of LXRβ in the peri-infarct area of rats housing in an enriched environment following pMCAO. Our data anticipate that enriched environment may have positive effects on lipid recycling in the ischemic hemisphere following experimental stroke.<strong></strong></p>

Stroke is the major cause of serious long-term disability with a sufficient acute treatment for only a very limited number of patients. Limited recovery of neurological functions occurs and can be elevated by a permissive post-stroke milieu. Housing animals in an enriched environment modulates regenerative mechanisms in the nonischemic peri-infarct area which might be an attractive target for pharmacological treatments to promote recovery. Upon ischemia, cellular lipids are released due to massive cell damage and free lipids significantly contribute to the progression of acute and delayed cell death. The aim of this study was to evalute the effect of enriched environment on lipid metabolism. In particular we characterize the activation of the transcription factor liver X receptor (LXR) in glial scar formation and regulation of cholesterol balance of relevance for functional recovery following stroke.                                      Brain tissues from animals subjected to permanent occlusion of middle cerebral artery (pMCAo) were analysed for LXRα and β protein expression. We found an upregulation and an increased transcriptional activity of LXRβ in the peri-infarct area of rats housing in an enriched environment following pMCAO. Our data anticipate that enriched environment may have positive effects on lipid recycling in the ischemic hemisphere following experimental stroke.