L’arthrose est une pathologie invalidante, caractérisée par une dégradation progressive du cartilage. L’usure n’est pas la seule caractéristique de l’arthrose qui implique d’autres modifications dans l’ensemble des tissus de l’articulation. Nos précédents travaux ont mis en évidence une accélération du renouvellement osseux dans l’os sous-chondral (l’os situé au contact direct du cartilage) qui se traduit par une dégradation osseuse précoce par les ostéoclastes suivi d’une condensation de l’os. Nous avons démontré que l’inhibition de la résorption protège de l’arthrose chez la souris. Les lésions arthrosiques sont prévenues par l’inhibition des ostéoclastes chez la souris, dont les médiateurs à effet anti-catabolique sont encore inconnus. Le but du travail était d’étudier le rôle de la sphingosine 1 phosphate (S1P), un médiateur lipidique du remodelage osseux produit par les ostéoclastes, sur le métabolisme chondrocytaire durant l’arthrose. Nous avons observé une augmentation de l’expression de S1P dans l’articulation arthrosique, notamment dans l’os sous-chondral et la synoviale. L’expression de SPHK1, l’enzyme majeure de la maturation de S1P est également augmentée dans le cartilage et l’os sous-chondral arthrosique murin et humain. In vitro, nous avons confirmé que l’activité de SPHK1 est augmentée au cours de la différenciation ostéoclastique. Nous avons donc évalué l’effet du sécretome ostéoclastique comme source de S1P sur le métabolisme chondrocytaire. Le sécretome ostéoclastique induit l’expression des métallo-protéases des cultures d’explants et de chondrocytes. L’expression des récepteurs 1 à 3 au S1P ainsi que la voie de signalisation MAPK associée sont activées. Seul le blocage du récepteur 2 (JTE-013 et siRNA) a permis de réduire l’augmentation des métallo-protéases initialement induite par le sécretome ostéoclastique. Le sécretome des ostéoclastes primaires isolée la souris LyM Cre-Sphk1-/- réduit le phénotype catabolique dans le chondrocyte alors que la délétion de SPHK1 chondrocytaire (Cre-lox recombinase in vitro) reste sans effets sur le métabolisme chondrocytaire induit par le sécretome ostéoclastique. In vivo, chez les souris LysM Cre-Sphk-/1 et les souris Col2 Cre-Sphk1-/-, le score arthrosique n’est pas changé. Ce résultat suggère que la délétion de Sphk1 dans le lignage myéloïde ou chondrocytaire n’est pas suffisante pour réduire le phénotype arthrosique. En revanche, la dégradation du cartilage est prévenue avec l’inhibition locale du S1PR2 et le blocage local de S1P avec un anticorps spécifique (le sphingomab). Nos résultats démontrent fortement l’implication du métabolisme de S1P dans la physiopathologie de l’arthrose. L’inhibition directe du S1P ainsi que sa signalisation via le récepteur 2 protège des lésions arthrosiques. Ces données suggèrent que Le ciblage de S1P pourrait être une piste thérapeutique pour l’arthrose. / High osteoclastogenesis accompanies early stages of osteoarthritis (OA). Cartilage loss is reduced when osteoclasts are inhibited in mice with bone hyperresorption. Although evidence showed that osteoclast-produced molecules affects chondrocyte metabolism, the mechanism by which inhibition of osteoclasts protects from cartilage damage is unclear. Our purpose was to investigate the role of Sphingosine 1 Phosphate, a lipid mediator secreted by osteoclasts known as mediator of bone remodeling, in chondrocyte metabolism and OA. We first observed that S1P expression is higher in subchondral bone cells of OA mice. In the same manner the both SPHK1 expression and activity is increased during in vitro osteoclast differentiation. Thus, we analyzed the effect of osteoclast secretome as the main source of S1P in chondrocyte catabolism. Osteoclasts secretome induced matrix degradation and metalloprotease expression (Mmp3, Mmp13) in femoral head explants and primary murine chondrocytes. The expression of S1P receptors 1-3 was increased in chondrocytes cultured with osteoclast secretome, as well as the activation of their signaling pathway (MAPK). However, only the inhibition of the receptor S1PR2 by JTE-013 and RNA silencing abolished the effect on MMP-3 and -13 in primary chondrocytes, but not those of S1PR1-R3. S1PR2 inhibition was confirmed in femoral head explants as JTE-013 reduced loss of proteoglycan and extracellular matrix degradation initially induced by osteoclast secretome. Furthermore, in vitro assay demonstrated that osteoclast secretome induced metalloprotease expression (MMP3, MMP13) in chondrocyte via MAPK (P38, JNK) pathway. To further investigate the role of S1P produced by the osteoclasts, we assessed the contribution of myeloid SPHK1, the main enzyme that metabolizes S1P. In vitro, secretome of LyM Cre-Sphk1-/- primary osteoclasts induced lower expression of Mmp3 in chondrocyte while not affected by SPHK1 deletion in chondrocytes (Cre-lox recombination in vitro). However, LysM Cre-Sphk1-/- mice and Col2 Cre-Sphk1-/- showed no reduce in osteoarthritis phenotype, suggesting the contribution of others cells in joint. However, targeting directly S1P by local administration of sphingomab or S1PR2 by JTE-013 showed a significant reduce in OA score compared to control. Our work shows that osteoclast secretome induce chondrocyte catabolism through the activation of S1P/S1PR2 signaling in chondrocytes. However, only the local inhibition of S1PR2 or S1P prevent against OA in mice. These data identify S1P as a therapeutic local target in OA.

Prevention of macrophage apoptosis in advanced atherosclerotic lesions can help stop atherosclerosis progression to vulnerable plaques. High density lipoprotein (HDL) can protect macrophages from apoptosis that has been induced by a variety of agents. We hypothesize that this is the consequence of the sphingolipid, sphingosine-1-phosphate (S1P), specifically carried by HDL, and transferred to S1P receptor 1 (S1P1) on the cells via the HDL receptor, scavenger receptor class B type 1 (SR-B1). Apoptosis was induced in murine peritoneal macrophages from wild type and different knockout mice with, tunicamycin, thapsigargin, staurosporine, or UV irradiation. Apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) or with cleaved caspase-3 (CC3) staining. Treatment of cells with HDL or S1P protected them against apoptosis induced by a variety of stimuli. In contrast, pre-treatment of HDL with S1P lyase, which irreversibly cleaves S1P, eliminated the ability of HDL to protect macrophages. Inhibition of SR-B1’s lipid transport activity reduced HDL dependant protection against apoptosis. Furthermore, HDL dependent protection against apoptosis induced by tunicamycin was prevented when the S1P receptor S1P1 was knocked out. However, this protection was not prevented when apoptosis was induced by staurosporine. These results suggest that the HDL mediated protection of macrophages against apoptosis is multi-faceted and one approach may involve SR-B1 mediated delivery of S1P from HDL to the S1P1. Understanding the mechanisms by which HDL elicits atheroprotective signalling in macrophages will provide insight into new targets for therapeutic intervention in atherosclerotic disease. / Thesis / Master of Science in Medical Sciences (MSMS) / Atherosclerosis, is a disease where in the artery walls thicken due to cholesterol build-up, is the major underlying cause for cardiovascular diseases, which is currently a leading cause of death in many populations. We believe that HDL, the “good” cholesterol and S1P, a small molecule carried by HDL, can help prevent the progress of atherosclerosis by preventing macrophages, cells that absorb the cholesterol, from dying. We attempt to prove this by providing S1P or HDL to macrophages that are made to undergo cell death. Results show that both HDL and S1P can protect cells against cell death induced by many factors. However, HDL can protect against certain cell death inducing stimuli without the need for S1P and more research is required to fully understand HDL’s protective role in atherosclerosis. Understanding how HDL elicits atheroprotective signalling in macrophages will help in finding new drugs and therapies to reduce atherosclerosis-based deaths across the world.

No description available.

In the human adrenal cortex, adrenocorticotropin (ACTH) activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondria and chronically up-regulating the transcription of steroidogenic genes (including CYP17). Sphingolipids are a diverse family of phospholipids and glycolipids that mediate a wide variety of cellular processes, including apoptosis, proliferation, and survival. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes that are involved in cholesterol biosynthesis and fatty acid metabolism. In this study, we investigated the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of several sphingolipid species was quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of sphingomyelin, ceramides, sphingosine (So) and sphingosine-1-phosphate (S1P). However, both ACTH and Bt2cAMP increased the activity of sphingosine kinase and the amounts of S1P released into the media. Both So and S1P increased CYP17 mRNA expression and increased cortisol biosynthesis. This increase in CYP17 transcription occurs by promoting SREBP binding to an SRE at -450/-436 basepairs upstream of the transcription initiation site. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that Bt2cAMP and S1P treatment results in an increase in acetylation of histone H3 and SREBP1 binding to CYP17 promoter. Additionally, transient transfection studies using wild type or mutated hCYP17 promoters and RNA interference (RNAi) assays confirmed the role of SREBP1 in mediating the stimulatory effect of S1P on CYP17 transcription. In summary, our studies demonstrate a link between sphingolipid metabolism and ACTH-dependent steroidogenesis which requires the activation of SREBP1 in human adrenal cortex.

Sickle cell disease (SCD) is a disorder in which defective hemoglobin causes sickling of red blood cells, inducing painful vaso-occlusive crises when blood flow is blocked at sites of red blood cell (RBC) clotting that can ultimately result in organ failure or death. This work demonstrates that sphingolipid metabolism is dysregulated in SCD and that this pathway can be targeted pharmacologically to prevent vaso-occlusion. We suggest a pathway in which the sickling of RBCs in SCD activates acid sphingomyelinase, altering the distribution and concentration of sphingolipids in the RBC membrane and resulting in the production of sphingolipid-rich microparticles that are secreted and can interact with cells in circulation. Sphingosine-1-phosphate (S1P) is believed to be a key modulator of SCD because it is stored at high concentrations in RBCs. Sphingolipid metabolism was confirmed to be dysregulated in SCD; most notably, S1P was significantly elevated in RBCs, and plasma, and microparticles, and the activity of acid sphingomyelinase and concentration of its byproduct, microparticles, were significantly elevated in SCD RBCs. Treatment of monocytes with S1P and SCD RBCs increased their adhesion over four-fold to endothelial cells, indicating that altered sphingolipid distribution in RBCs may contribute to vaso-occlusion through increasing myeloid cell adhesion. A cytokine profile of macrophages treated with SCD microparticles suggest that microparticles play a role in this process by increasing the secretion of inflammatory cytokines associated with SCD crises, including MIP-1α, IL-6, and TNF-α. Pilot in vitro studies in RBCs and in vivo studies in mice implicate that drugs targeting the sphingolipid metabolic pathway may be more effective treatment options than blood transfusions in managing SCD and preventing vaso-occlusive crises.

Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR). Metabolomic profiling of placentas from such pregnancies has identified deranged sphingolipid metabolism as one of only a handful of pathways altered in PE/FGR. In other systems, the bioactive sphingolipid, sphingosine-1-phosphate (S1P) controls cell migration therefore this study aimed to determine its effect on extravillous trophoblast (EVT) function. S1P (50 nM–10 µM) attenuated the migration of the EVT cell lines, Swan-71 and SGHPL-4 (n = 6; p < 0.05) and also the outgrowth of trophoblast from explants of human first trimester placenta. Quantitative PCR and immunolocalisation studies demonstrated that both EVT cell lines and primary EVT express S1P receptors 1, 2 and 3 in similar abundance. Receptor inhibitors were used to reveal S1PR2 as the receptor responsible for mediating the inhibitory effect of S1P inhibitory effect; JTE-013 (100 nM) a specific S1PR2 inhibitor, abolished S1P- attenuated migration (n = 6; p < 0.05 versus S1P alone) whereas treatment with the S1PR1/3 inhibitor, FTY720 (100 nM; n = 6) had no effect on S1P activity. Ligand binding to S1PR2 can activate numerous intracellular signalling pathways via receptor association with the G proteins, Gα12/13, Gαq or Gαi; however, analysis of Swan-71 cell migration and actin cytoskeleton in the presence of S1P ± the Rho kinase inhibitor, Y-27632 (10 µM; n = 6) suggested preferential activation of Gα12/13. Nonetheless, S1P does activate Gαi in Swan-71 cells, as demonstrated by analysis of cAMP levels and phosphorylation of downstream signalling molecules; however attempts to shift the balance of intracellular pathway activation towards Gαi/Rac using siRNA-mediated knockdown of the Rac inhibitor ARGHGAP22 did not attenuate S1P inhibition of cellular motility, Subsequent experiments explored the possibility of preventing S1P’s actions by modulating EVT S1P receptor isoform expression using factors, including hormones and oxygen, previously reported to affect trophoblast migration or the expression of S1PR in other systems. Neither EGF nor low oxygen levels influenced S1PR expression however both IGF-II (10nM; p<0.05) and vitamin D (10nM; p<0.05) prevented the inhibitory effect of S1P on Swan-71 cell migration, the latter as a result of a significant reduction in S1PR2 expression (4-fold decrease; p<0.05).This study demonstrates that, although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2 / Gα12/13 to activate Rho and actin stress fibre formation and thereby acts as potent inhibitor of EVT migration. Strategies aimed at shifting the balance of receptor isoform expression, may provide a mechanism for improving impaired trophoblast migration in compromised pregnancies. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Thus these data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

Sterile inflammation is a key component of a variety of diseases including, gout, arthritis, type 1 diabetes, Alzheimer’s disease and multiple sclerosis (MS). Sterile inflammation induces the recruitment of immune cells via chemokines, such as CCL5 and CXCL10. Expression of these chemokines is dependent on IRF-1. Recently the FDA has approved the use of a pro-drug, FTY720 that after phosphorylation becomes a S1P mimetic for the treatment of MS. This report describes two novel and opposing mechanisms of S1P action in sterile inflammation. First, intracellular S1P acts as a cofactor of cIAP2 that inducesIL-1-dependent K63-polyubiquitination of IRF-1, which leads to the recruitment of immune cells to the site of inflammation. Conversely, extracellular S1P provides a feedback loop that inhibits CXCL10 and CCL5 expression through S1PR2 signaling. Accordingly, immune cell infiltration to sites of sterile inflammation is increased in S1PR2-/- production via calcium-dependent, but cAMP- and PKA-independent mechanisms that likely involve c-Fos expression and unconventional PKC activation. Elevated c-Fos could competitively inhibit CCL5 expression directly or indirectly via blocking IFN production. These two novel pathways highlight unexpected aspects of S1P signaling, and provide potential mechanisms that can be exploited for the improvement of therapeutics for the treatment of MS.

Sphingosine 1-phosphate (S1P) receptor (S1PR) modulators have a complex mechanism of action, which are among the most efficient therapeutic options in multiple sclerosis (MS) and represent a promising approach for other immune-mediated diseases. The S1P signaling pathway involves the activation of five extracellular S1PR subtypes (S1PR1–S1PR5) that are ubiquitous and have a wide range of effects. Besides the immunomodulatory beneficial outcome in MS, S1P signaling regulates the cardiovascular function via S1PR1–S1PR3 subtypes, which reside on cardiac myocytes, endothelial, and vascular smooth muscle cells. In our review, we describe the mechanisms and clinical effects of S1PR modulators on the cardiovascular system. In the past, mostly short-term effects of S1PR modulators on the cardiovascular system have been studied, while data on long-term effects still need to be investigated. Immediate effects detected after treatment initiation are due to parasympathetic overactivation. In contrast, long-term effects may arise from a shift of the autonomic regulation toward sympathetic predominance along with S1PR1 downregulation. A mild increase in blood pressure has been reported in long-term studies, as well as decreased baroreflex sensitivity. In most studies, sustained hypertension was found to represent a significant adverse event related to treatment. The shift in the autonomic control and blood pressure values could not be just a consequence of disease progression but also related to S1PR modulation. Reduced cardiac autonomic activation and decreased heart rate variability during the long-term treatment with S1PR modulators may increase the risk for subsequent cardiac events. For second-generation S1PR modulators, this observation has to be confirmed in further studies with longer follow-ups. The periodic surveillance of cardiovascular function and detection of any cardiac autonomic dysfunction can help predict cardiac outcomes not only after the first dose but also throughout treatment.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Résumé confidentiel / Confidential abstract