Hedgehog signaling is a pathway essential in embryonic development, adult stem cell maintenance, and is implicated in the formation and progression of cancer. Signaling in this pathway is triggered when the secreted protein Hedgehog binds to its membrane receptor, Patched. Patched normally inhibits the seven-spanner transmembrane protein Smoothened (Smo). Binding of Hedgehog inhibits Patched resulting in Smo derepression. Active Smo then triggers the activation of the cytoplasmic steps of the signaling pathway.

O estresse oxidativo pode desempenhar um papel importante na etiologia das complicações no diabetes mellitus. O aumento da produção de espécies oxidantes promove modificações em moléculas endógenas, incluindo o colesterol. Os óxidos de colesterol (Cox) são formados a partir da oxidação do colesterol, por processos enzimáticos e por processos mediados por radicais livres, apresentando importantes efeitos biológicos que podem contribuir para o desenvolvimento do processo aterosclerótico no diabetes. Nesse estudo determinou-se as concentrações dos Cox, em pacientes diabéticos e indivíduos intolerantes à glicose, para estabelecer se os COx são marcadores sensíveis da lipoperoxidação na intolerância à glicose e no diabetes As concentrações plasmáticas dos COx foram determinadas por GC-FID nos seguintes grupos: diabéticos tipo 1 (DM1), diabéticos tipo 2 (DM2), intolerantes à glicose (IGT) e normoglicêmicos (controles). As concentrações dos óxidos de colesterol totais foram mais elevadas nos grupos DM1 e DM2 em relação aos controles normoglicêmicos (p<0,05). As concentrações plasmáticas do 7&#945;- hidroxicolesterol (7&#945;-OH), 7&#946;-hidroxicolesterol (7&#946;-OH) e 25- hidroxicolesterol (25-OH) foram mais elevadas no grupo DM1 comparado ao grupo DM2 (p<0.05). A comparação entre os grupos controle, IGT e DM 2 indicou aumento significativo das concentrações de 7&#946;-OH, colesterol-&#946;- epóxido e colesterol-&#945;-epóxido no grupo DM 2 (p<0.05). Portanto, os óxidos de colesterol podem ser considerados como um biomarcador sensível da lipoperoxidação para indicar a intensidade de modificação oxidativa dos lípides em pacientes diabéticos. / Oxidative stress can play an important role in the etiology of the complications of diabetes mellitus. The increase in the production of oxidant species promotes alterations in endogenous molecules, including cholesterol. Cholesterol oxides (COx) are formed by the oxidation of cholesterol by enzymatic processes or by processes involving free radicals. They present important biological effects that can contribute to the development of the atherosclerotic process in diabetes. In this study, the concentrations of the COx in diabetic patients and individuals who are intolerant to glucose was determined in order to establish whether the Cox are sensitive markers of lipoperoxidation in glucose intolerance and diabetes. Serum concentrations of the COx were determined by GC-FID in the following groups: Type 1 diabetics (DM1), type 2 diabetics (DM2), patients intolerant to glucose (IGT) and normoglycemic subjects (controls). The concentrations of total cholesterol oxides were found to be elevated in the DM1 and DM2 groups with respect to the normoglycemic subjects (p<0.05). The serum concentrations of 7&#9465- hydroxicholesterol (7&#945;-OH), 7&#946;-hydroxicholesterol (7&#$946;-OH) and 25-hydroxicholesterol (25-OH) were found to be increased in the DM1 group with respect to the DM2 group (p<0.05). The comparison between the control, IGT and DM2 groups indicated a significant increase in the concentrations of 7&#946;-OH, cholesterol-&#946;-epoxide and cholesterol-&#945;-epoxide in the DM2 group (p<0.05). In conclusion, cholesterol oxides could serve as suitable biomarker of lipoperoxidation to indicate the intensity of lipid oxidative mofications in diabetic patients.

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

The presence of apoptotic cells in atherosclerotic lesions has been broadly reported in the past ten years. The majority of these apoptotic cells are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains to be elucidated. Elevated expression of Bax, one of the pivotal pro-apoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol treated mouse macrophages. In this study, we evaluated the influence of Bax deficiency on apoptosis in macrophage-like P388D1 cells by using small interfering RNA (siRNA) to suppress Bax expression, as well as in peritoneal macrophages isolated from Bax null mice (Bax-/-). Apoptotic activities in both cell types deficient for Bax were significantly reduced compared to that in control cells. To examine the effect of macrophage Bax deficiency on the development of atherosclerosis, fourteen 8-week-old male LDL-receptor null (LDLR-/-) mice were lethally irradiated and reconstituted with either wild type (WT) C57BL6 or Bax-/- bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax-/- group. However, quantitation of cross sections from proximal aortas revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax-/- group compared to the WT group. A 53% decrease in apoptotic macrophages in the Bax-/- group was found by TUNEL staining (P<0.05). In conclusion, Bax deficiency produces a reduction of apoptotic activity in macrophages and is associated with the accelerated atherosclerosis in LDLR null mice fed a Western diet. These results strongly support our hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.

Oxidized low-density lipoproteins (OxLDL) mediate many of the pathological events associated with atherosclerosis. Oxysterols, the major cytotoxic component of OxLDL, induce apoptosis in macrophages by a calcium flux mediated activation of cytosolic phospholipase A2 resulting in the release of arachidonic acid (AA). Inhibition of AA metabolism has been shown to protect macrophages from oxysterol-induced apoptosis. The current study explores the steps in the oxysterol-induced apoptosis signaling pathway in murine macrophages subsequent to the liberation of AA. To elucidate this mechanism, two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol (25-OHC), were used to induce apoptosis in murine macrophage cell lines (P388D1, and Raw 264.7) and mouse peritoneal macrophages (MPMs). Pharmacological inhibition of eicosanoid synthesis or genetic knockout of important eicosanoid biosynthetic genes had no significant effect on the induction of apoptosis by oxysterols in macrophages. The induction of apoptosis in macrophage cell lines and MPMs by oxysterols and OxLDL was suppressed by Sandoz 58-035, an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT). Furthermore, in comparison to wild-type MPMs, ACAT-1 deficient MPMs were found to be resistant to apoptosis induced by oxysterols or OxLDL. Macrophages treated with 7KC accumulated ACAT-derived cholesteryl and 7-ketocholesteryl esters. An inhibitor of cholesterol trafficking, U18666A, specifically prevented the accumulation of cholesteryl esters, but not 7-ketocholesteryl esters nor the induction of apoptosis. An inhibitor of cPLA2 prevented the accumulation of 7-ketocholesteryl esters. This inhibition was reversed by supplementing oleic acid or AA; however, only AA supplementation restored the induction of apoptosis. These results suggest that oxysterols not only initiate the apoptosis pathway by activating cPLA2, but also participate in the downstream signaling pathway when esterified by ACAT to form arachidonyl oxysterols. We also demonstrate that macrophages lacking the cannabinoid type-2 (CB2) receptor have reduced levels of apoptosis when treated with oxysterols or OxLDL in comparison to wild-type macrophages and that a CB2 specific antagonist blocks oxysterol-induced apoptosis in macrophages suggesting that the CB2 receptor is involved in this pathway, perhaps by interacting with the arachidonyl oxysterols.

L’infarctus du myocarde représente un problème de santé publique dont le traitement de choix consiste à restaurer le flux sanguin (reperfusion) à travers le tissu ischémié dans les plus brefs délais, cependant cette procédure s’accompagne de lésions supplémentaires dont les mécanismes ne sont pas totalement connus. Notre laboratoire a observé que la reperfusion du myocarde s’accompagne d’une accumulation de cholestérol et d’oxystérols dans les mitochondries qui sont impliquées dans l’apparition de ces lésions. L’objectif de ce projet de thèse a donc été d’identifier les mécanismes responsables de cette accumulation. L’utilisation de stratégies cardioprotectrices (statines, exercice physique, ligand de la protéine translocatrice (TSPO)), visant à réduire la concentration cellulaire ou mitochondriale de cholestérol ou le stress oxydant, a tout d’abord permis de mettre en évidence une relation entre l’effet cardioprotecteur et l’inhibition de l’accumulation des stérols mitochondriaux. Cette relation persiste en présence d’une hypercholestérolémie. Par ailleurs, nous avons montré que l’accumulation mitochondriale de cholestérol et d’oxystérols est due à la translocation de la protéine StAR (Steroidogenic Acute Regulatory protein) du cytosol à la mitochondrie dans les premières minutes de la reperfusion et que ce phénomène est modulé par le TSPO. L’inhibition de l’expression mitochondriale de la protéine StAR pourrait représenter une stratégie intéressante afin de protéger le myocarde ischémié de la reperfusion et ce notamment en cas d’hypercholestérolémie. / Myocardial infarction represents a serious public health issue which requires the restoration of the blood flow (reperfusion) in the ischemic tissue as soon as possible; however cardiac reperfusion also induces additional injuries whose mechanisms are not completely established. Our laboratory showed that myocardial infarction induces the accumulation of cholesterol and oxysterols in the mitochondria which are involved in the induction of reperfusion injury. The objective of this thesis project was to identify the mechanisms responsible for this accumulation. The use of cardioprotective strategies (statin, physical exercise, translocator protein (TSPO) ligand), aiming at reducing the cellular and mitochondrial cholesterol concentrations or the oxidative stress, showed the relation between the cardioprotective effect and the inhibition of the mitochondrial sterol accumulation. This relation persists in hypercholesterolemic animals. Furthermore, we showed that the mitochondrial cholesterol and oxysterol accumulation is caused by the translocation of StAR (Steroidogenic Acute Regulatory protein) from cytosol to mitochondria during the first minutes of the reperfusion and this phenomenon is regulated by the TSPO. The inhibition of the mitochondrial expression of StAR could be an interesting approach in order to protect the ischemic myocardium from reperfusion injury, especially in a hypercholesterolemic context.

<p> The liver X receptors (LXRs) are members of the nuclear hormone receptor (NHR) superfamily of transcription factors and are activated by oxysterols. As such, LXRs act as "cholesterol sensors" and play an integral role in cholesterol homeostasis by modulating the expression of genes involved in lipid transport and metabolism as well as inflammation. </p> <p> This thesis begins by describing the modulation of LXR transactivation by PKC. Specifically, transactivation by LXRα is decreased upon activation of PKC signalling pathways as assessed by LXR reporter gene analysis and endogenous target gene expression. These findings reveal a mode of regulation of LXRα that may be relevant to disease conditions where aberrant PKC signalling is observed. </p> <p> The second and third part of the thesis turns the attention to the role of LXR in human airway smooth muscle (hASM), a crucial effector cell in asthma progression. For the first time, research described here indicates that primary human ASM cells express functional LXRs. Moreover, LXR target genes ABCA 1 and ABCG I were highly induced upon the addition of LXR agonists leading to enhanced cholesterol efflux to apoAI and HDL, a process dependant entirely on ABCA I. Furthermore, activation of LXR inhibited the expression of multiple cytokines in response to inflammatory mediators and inhibited the proliferation and migration of hASM cells, two important processes that contribute to the airway remodelling observed in the asthmatic lung. </p> <p> This body of work suggests that modulation of LXR offers prospects for new therapeutic approaches in the treatment of asthma. Furthermore, it establishes a critical role for ABCA 1 in lipid transport in ASM cells and suggests that dysregulation of cholesterol homeostasis in these cells may be important. These findings have broad implications in the association of hypercholesterolemia and AHR and places LXR at the forefront of novel therapeutic avenues to treat inflammatory lung disease. </p> / Thesis / Doctor of Philosophy (PhD)

Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators. The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS. It was found that aged rats exhibited a significant increase in brain AA and decrease in Σn-3 and Σn-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice. These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease.

Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators. The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS. It was found that aged rats exhibited a significant increase in brain AA and decrease in ¿n-3 and ¿n-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice. These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease. / Libyan Government

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