Rôle de la voie sphingomyelinase neutre de type 2 - sphingosine kinase 1 - sphingosine-1-phosphate dans l'effet angiogénique des LDL oxydées

L'athérosclérose et ses complications cardiovasculaires représentent la principale cause de morbi-mortalité dans les pays développés et posent un problème majeur de santé publique. De nombreux facteurs de risque, ont été décrits, en particulier l'hypercholestérolémie liée aux LDL circulantes. L'oxydation de ces LDL constitue une étape déterminante dans le processus d'athérogénèse. Dans la paroi des artères de moyen et gros calibre, on observe un réseau microvasculaire (les vasa vasorum) localisé au niveau de l'adventice et qui a pour rôle d'apporter oxygène et nutriments aux couches les plus externes de la paroi artérielle. Alors que ces microcapillaires ne sont pas retrouvés au niveau de la media et de l'intima des artères normales, il se produit une neovascularisation en regard de l'hyperplasie intimale des artères athéroscléreuses. Les LDL oxydées, par leur effet proangiogénique, peuvent contribuer à cette neovascularisation qui participerait à l'expansion de la plaque mais augmenterait aussi le risque d'hémorragies intraplaques ainsi que le risque de rupture et d'accidents athérothrombogènes. Le mécanisme angiogénique des LDLox est peu connu, et l'implication de la sphingosine-1-phosphate (S1P), un second messager de la voie des sphingolipides, impliqué dans l'angiogenèse, n'a pas été rapporté dans l'angiogenèse induite par les LDLox. Le but de ce travail est d'évaluer l'implication et les mécanismes d'activation de la voie des sphingolipides, avec la génération consécutive de S1P, dans l'effet pro-angiogénique des LDL oxydées sur les cellules endothéliales microvasculaires. Dans une première partie nous avons montré un effet biphasique des LDL oxydées sur les HMEC-1 (Human Microvascular Endothelial Cells), avec un effet proangiogénique à faibles doses (20 à 50µg/ml) et une absence d'angiogenèse pour des doses plus élevées non encore toxiques (100 µg/ml). Au-delà de ces doses, nous observons un effet toxique, comme cela a déjà été précédemment décrit. L'inhibition par siRNA de l'expression de SK1 (sphingosine kinase-1 impliquée dans la génération de S1P) et l'utilisation d'un anticorps bloquant de la S1P (Sphingomab(tm)), permettent d'inhiber efficacement la tubulogenèse des cellules endothéliales induite par les LDLox in vitro. L'angiogenèse chez la souris C57/Bl6 (étudiée par Matrigel plugs contenant des LDL oxydées), est efficacement prévenue par l'administration de l'anticorps bloquant Sphingomab(tm), définissant le rôle majeur de ce lipide bioactif dans le signal angiogénique des LDLox in vitro et in vivo. Sur un plan mécanistique, l'activation de SK1 et l'angiogenèse induite par les LDLox sont bloquées par l'utilisation d'anticorps bloquants anti-CD36 et anti-LOX-1 montrant l'implication des récepteurs scavengers des LDLox dans cette signalisation. L'utilisation d'un inhibiteur de l'activité kinase du VEGFR2 bloque dans une même proportion l'angiogenèse induite par les LDLox et par de la S1P exogène, évoquant une transactivation du VEGFR par les récepteurs membranaires de la S1P, en l'absence de synthèse de VEGF par les HMEC-1 sous l'effet des LDLox. Dans une deuxième partie nous avons montré que la génération de S1P consécutive à l'activation de la SK1 sous l'effet des LDLox, est étroitement liée à l'activation en amont de la nSMase2 dans des cellules endothéliales in vitro, impliquant également le stress oxydant et p38MAPK. L'utilisation d'un inhibiteur chimique de la nSMase2 bloque efficacement l'angiogénèse induite par les LDLox in vitro, ainsi que l'activation de SK1. Le time-course de génération de ROS et de phosphorylation de p38 dans les HMEC-1 soumises à des doses angiogéniques de LDLox est plus précoce que l'activation des enzymes de la voie des sphingolipides. L'utilisation des antioxydants NAC et trolox ainsi qu'un inhibiteur de phospho-p38MPAK bloquent à la fois la tubulogenèse et l'activation de nSMase2 et de SK1 in vitro, évoquant leur implication dans cette signalisation en amont de la voie des sphingolipides. Dans une troisième partie nous avons étudié le lien entre l'activation de la voie des sphingolipides par les LDLox et la connexine-43, protéine constitutive des jonctions gap permettant la transduction d'une signalisation entre cellules adjacentes. Grâce à l'utilisation d'inhibiteurs pharmacologiques des connexines, nous avons obtenu une inhibition efficace de l'activation de la SK1 et de la tubulogenèse induite par les LDLox in vitro, permettant d'évoquer la participation des jonctions gap dans la transduction du signal angiogène des LDLox impliquant la génération de S1P. En prespective à ce travail, nous projetons d'approfondir les mécanismes impliquant la connexine-43 dans cette signalisation notamment par rapport à la nSMase2 et à p38MPAK et d'établir un lien avec le développement de la vascularisation des plaque in vivo à l'aide de marquage de SK1 ou connexine-43 sur des échantillons d'artériectomie carotidienne. Ces travaux mettent en évidence de nouvelles cibles (S1P, connexine-43) impliquées dans le mécanisme de la néoangiogenèse intraplaque, évoquant de nouvelles perspectives thérapeutiques pour limiter les complications athérothrombogènes grèvant de façon importante le pronostic des patients atteints d'athérosclérose.Atherosclerosis and relative complications represent the main cause of morbi-mortality in western countries and arise a major problem for public health. Many risk factors have been described, in particular hypercholesterolemia linked to circulating LDL. The oxidation of LDL constitutes a decisive stage in atherogenesis. The vascular wall of the medium and large arteries is vascularized by a microcapilaries plexus (vasa vasorum) located in adventice, and in charge to bring oxygen and nutrients to the most external layers of the arterial wall. Although, no capillaries are found in the intima or media of normal arteries, we observe neovascularization under intimal hyperplasia of atherosclerotic arteries. As oxidized LDL could display angiogenic properties, they may contribute to this neovascularization, taking part in plaque growth but also in increasing risk of intraplaque heamorrage and destabilization leading to atherothrombotic complications. The angiogenic mechanisms of oxidized LDL is yet incompletely known, and the involvement of sphingosine-1-phosphate (S1P), a second messenger of sphingolipid pathway, well known for its angiogenic properties, has never been reported in oxLDL-induced angiogenesis. The aim of this work is to evaluate the involvement of sphingolipid pathway and its mechanisms of activation, with subsequent generation of S1P, in proangiogenic effects of oxidized LDL on microvascular endothelial cells. In a first part, we have demonstrated a biphasic effect exhibited by oxidized LDL in HMEC-1 (Human Microvascular Endothelial Cells), low oxLDL concentrations (20 to 50µg/ml) being angiogenic, whereas higher concentrations being not toxic yet (100µg/ml) are non angiogenic. Beyond these concentrations, as expected, a toxic effect was observed. Inhibition of SK1 (sphingosine kinase-1) expression by using a specific SiRNA, and use of S1P blocking antibody (Sphingomab(tm)), exibit an efficient inhibition of oxLDL-induced tubulogenesis of endothelial cells in vitro. Angiogenesis in C57/Bl6 mice (using Matrigel plug assay containing oxLDL), is efficiently prevented by administration of the S1P blocking antibody Sphingomab(tm), defining the major role of this bioactive lipid in the oxLDL angiogenic signal, in vitro and in vivo. Mechanisticaly speaking, ox-LDL-induced SK1 activation and angiogenesis are blocked by using anti-CD36 and anti-LOX-1 blocking antibody, showing the involvement of oxLDL scavenger receptors in this signaling. The use of a sphingosine kinase inhibitor of VEGFR2 blocks ox-LDL-induced angiogenesis in the same range as exogenous S1P-induced angiogenesis, evoking a cross-talk between VEGFR and membranary S1P receptors, in the absence of ox-LDL-induced VEGF synthesis in HMEC-1. In a second part, we have shown that ox-LDL-induced SK1 activation and subsequent S1P synthesis, was tightly linked to the amoung nSMase2 activation in endothelial cells in vitro, involving also oxidative stress and p38MPAK. The use of a chemical inhibitor of nSMase2 efficiently blocks both ox-LDL-induced angiogenesis and SK1 activation in vitro. The time course of ROS generation and p38MAPK phosphorylation in HMEC-1 submitted to angiogenic doses of oxLDL appears earlier than enzymes activation of sphingolipid pathway. Using antioxydants as NAC and trolox, as well as a phospho-p38MPAK inhibitor, we block both angiogenesis and nSMase2 and SK1 activation in vitro, evoking implication of oxidative stress and p38MPAK in this signalling amoung of sphingolipid pathway. In a third part, we have studied the link between oxLDL induction of sphingolipid pathway and connexine-43, a constitutive protein of gap junctions allowing signal transduction between adjacent cells. Through the use of two connexine pharmacological inhibitors, we have obtained an efficient inhibition of oxLDL-induced SK1 activation and tubulogenesis in vitro, evoking that gap junctions may take part in oxLDL angiogenic signalling, implicating S1P generation. In the perspective of this work, we plan to intensify the knowlegde of mechanisms involving connexine-43 in this signaling, particularly in relation to sphingolipid pathway and p38MAPK. Thereafter we would like to establish a link between this work and the developement of plaque neovascularization in vivo, in support of SK1 or connexines-43 staining on endarterectomy samples of human carotids from patients. These results arise new targets (S1P, connexine-43) implicated in development of intraplaque neoangiogenesis, and new therapeutic possibilities to limit atherothrombotic complications that significantly worsen the prognosis of atherosclerotic patients

Scenario of Accelerating Universe from the Phenomenological \Lambda- Models

Dark matter, the major component of the matter content of the Universe, played a significant role at early stages during structure formation. But at present the Universe is dark energy dominated as well as accelerating. Here, the presence of dark energy has been established by including a time-dependent $\Lambda$ term in the Einstein's field equations. This model is compatible with the idea of an accelerating Universe so far as the value of the deceleration parameter is concerned. Possibility of a change in sign of the deceleration parameter is also discussed. The impact of considering the speed of light as variable in the field equations has also been investigated by using a well known time-dependent $\Lambda$ model.Comment: Latex, 9 pages, Major change

Future directions for therapeutic strategies in post-ischaemic vascularization: a position paper from European Society of Cardiology Working Group on Atherosclerosis and Vascular Biology

Modulation of vessel growth holds great promise for treatment of cardiovascular disease. Strategies to promote vascularization can potentially restore function in ischaemic tissues. On the other hand, plaque neovascularization has been shown to associate with vulnerable plaque phenotypes and adverse events. The current lack of clinical success in regulating vascularization illustrates the complexity of the vascularization process, which involves a delicate balance between pro- and anti-angiogenic regulators and effectors. This is compounded by limitations in the models used to study vascularization that do not reflect the eventual clinical target population. Nevertheless, there is a large body of evidence that validate the importance of angiogenesis as a therapeutic concept. The overall aim of this Position Paper of the ESC Working Group of Atherosclerosis and Vascular biology is to provide guidance for the next steps to be taken from pre-clinical studies on vascularization towards clinical application. To this end, the current state of knowledge in terms of therapeutic strategies for targeting vascularization in post-ischaemic disease is reviewed and discussed. A consensus statement is provided on how to optimize vascularization studies for the identification of suitable targets, the use of animal models of disease, and the analysis of novel delivery methods

Studying Green Purchase Behavior Of Malaysian Consumers

This study has used theory of planned behaviour to study green purchase behaviour of Malaysian consumer. This theory uses perceived consumer effectiveness, arritudes and subjective norms as predisctors of behavioural intention

Multicriteria decision evaluation of adaptation strategies for vulnerable coastal communities

According to the IPCC (2007) fourth assessment report, small islands and coastal communities have a set of characteristics that makes them very vulnerable to climate change impacts, mainly sea-level rise and storm surges. Coastal hazards including inundation, salinisation of the water supply, and erosion threaten vital infrastructure that support coastal communities. Although Canada has the longest coastline in the world, little work has been done on impacts of climate change and adaptation to these impacts in the Canadian coastal zones. This research is part of an International Community-University Research Alliance (ICURA) C-Change, project to develop a multicriteria decision evaluation and support for the systems analysis of adaptation options for coastal communities toward adapting to environmental changes. This study estimates the vulnerability of coastal communities with respect to their environmental, economic, social, and cultural dimensions. It also applies a group version of the Analytical Hierarchy Process for identifying decisions that various stakeholders make on suggested adaptation strategies. This study develops a methodological framework that is applicable to various coastal and small island contexts. The application of the proposed framework is further discussed in a case study conducted on the communities of Charlottetown, Prince Edward Island (PEI), and Little Anse on Isle Madame, Nova Scotia. Specifically, the state of the Little Anse breakwater is analyzed and new adaptation options are presented and evaluated. This research has illustrated and applied a process of decision evaluation and support that explicitly engages multiple participants and critieria in complex problems situations involving environmental change in coastal communities.Master's degre

La contraception en médecine générale

Le but de ce travail est de réaliser un descriptif des moyens de contraception actuellement disponibles en France. Le travail principal fut un travail bibliographique et d'analyse des informations obtenues. Il en ressort une grande diversité de moyens contraceptifs, adaptables à chaque patiente en fonction de ses demandes, besoins ou contraintes physiologiques voire pathologiques. Cette étude permet la rédaction d'un guide pratique pour le médecin généraliste dans son travail quotidien.TOULOUSE3-BU Santé-Centrale (315552105) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Angiogenesis in the atherosclerotic plaque

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Shaping Approach Responses as Intervention for Specific Phobia in a Child with Autism

We evaluated contact desensitization (reinforcing approach responses) as intervention for specific phobia with a child diagnosed with autism. During hospital-based intervention, the boy was able to encounter previously avoided stimuli. Parental report suggested that results were maintained postdischarge

Dual signaling evoked by oxidized LDLs in vascular cells

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Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody

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