Etude du rôle du stress métabolique hyperlipidique dans le développement des pathologies cardiovasculaires par approches transcriptomique et protéomique

Les maladies cardiovasculaires (MCV) représentent aujourd'hui une des 1ères causes de mortalité dans le monde. De nombreux facteurs sont incriminés dans le développement de ces pathologies, dont l'hyperlipémie, l'insulino-résistance, l'obésité viscérale ou encore l'hypertension, eux-mêmes fortement dépendants de mauvaises habitudes alimentaires. Aussi, l'association d'une consommation importante de graises saturées avec la survenue des MCV est aujourd'hui bien établie. La pathogenèse de ces maladies, particulièrement l'athérothrombose, a fait l'objet d'une recherche intensive. L'implication du stress oxydant et de l'inflammation a été soulignée, et a servi de base à de nouvelles stratégies thérapeutiques. Si la connaissance moléculaire de l'athérothrombose est aujourd'hui relativement bien établie, peu de données sont disponibles concernant les stades précoces de la pathogenèse. Le stress métabolique, consécutif à une consommation importante de graisses, constitue un de ces stades. Ce stress a été observé lors de la période postprandiale hyperlipidique. En effet, une charge lipidique résulte en un dysfonctionnement vasculaire et à l'élévation plasmatique des marqueurs du stress oxydant et de l'inflammation chez le volontaire sain. Premièrement, dans le but de mieux caractériser le stress métabolique en réponse à la consommation des lipides, l'influence d'une consommation de graisses saturées pendant un temps court de 3 semaines a été exporée sur le modèle murin. Nous avons montré que ce temps court de régime était suffisant à induire des perturbations métaboliques (hypercholestérolémie et hyperglycémie). En parallèle de ces variations métaboliques, certaines régulations d'expression de gènes clefs du métabolisme lipidique ont été identifiées au niveau hépatique. Parmi elles, la surexpression du gène de la "sérum amyloid A", se traduisant par l'augmentation de sa concentration plasmatique, suggèrent l'installation des processus de l'inflammation suite au régime hyperlipidique. L'impact de ce régime a ensuite été exploré au niveau vasculaire, par l'analyse des profils transcriptomiques desaortes de ces animaux. Cette étude a révélé une réponse majeure des gènes du cytosquelette et de la matrice extracellulaire, et suggère un remodelage du tissu vasculaire suite à la consommation des graisses. Deuxièmement, le stress métabolique induit lors de la période postprandiale hyperlipidique a été étudié chez l'homme sain, par analyse protéomique du plasma prélevé suite à un repas riche en graisses. Cette approche a permis de démontrer l'importance de variations posttraductionnelles lors de la période postprandiale, notamment au niveau de protéines du complément (C3, C4 et ficolline 3) et du système hémostatique (fibrinogène). La dysfonction postprandiale de l'endothélium vasculaire a ensuite été explorée sur un modèle ex vivo, par une approche transcriptomique. Les principaux résultats de cette étude montrent que le sérum postprandial hyperlipidique exerce un effet antiprolifératif sur les cellules endothéliales vasculaires et entraîne des variations d'expression de gènes en faveur de l'arrêt du cycle cellulaire et de l'induction de l'apoptose dans ces cellules. Ces résultats sont en accord avec un effet délétère de la période postprandiale hyperlipidique sur l'endothélium vasculaire, et apportent de nouvelles données sur les mécanismes moléculaires impliqués. En conclusion, notre travail a permis d'identifier de nouveaux mécanismes d'un stress métabolique consécutif à une consommation importante de graisses.Cardiovascular diseases (CVD) are one of the leading causes of death in the world. A great number of risk factors are involved in the etiology of these pathologies, including insulin-resistance, hyperlipæmia, visceral obesity and hypertension. All of these could be at least in part related to unhealthy alimentary habits. The association of high saturated fat consumption with the occurence of CVD is now well established. Pathogenic mechanisms of these diseases, particularly atherothrombosis, have been the subject of intensive research. The involvement of oxidative stress and inflammatory processes are now well established. This served as the basis for new therapeutic strategies. If knowledge of the molecular processes involved in atherothrombosis is now fairly well known, little information is available regarding the early stages of the pathogenesis. The metabolic stress, due to high consumption of fat, is one of those stages. This stress has recently been identified during postprandial hyperlipæmia. Indeed, vascular dysfunction and elevated concentrations of biomarkers of oxidative stress and inflammation have been observed after a lipid challenge in healthy men. First, in order to characterize more precisely the metabolic stress, the influence of a high-fat diet for a short period of time of 3 weeks has been explored in a mouse model. We have shown that this short time of high fat consumption was sufficient to establish metabolic disturbances (hypercholesterolemia and hyperglycæ mia). In parallel to these metabolic changes, regulation of expression of some key genes involved in lipid metabolism was studied in liver. Among them, overexpression of the gene coding for "serum amyloid A" and resulting increase of its plasma protein concentration, suggest the induction of inflammatory processes following the 3 weeks high-fat diet. After that, the transcriptomic analysis of mice aortas was performed. This study revealed a broad response of extracellular matrix and cytoskeleton genes, and suggests vascular tissue remodelling following high fat consumption. Secondly, the metabolic stress induced during the postprandial hyperlipæmic state was studied by a proteomic analysis of plasma withdrawn after a high-fat challenge in healthy men. The obtained results have underlined the importance of post-translational modifications during the postprandial period, especially in the case of complement (C3, C4 anf ficollin 3) and hemostatic system (fibrinogen) proteins. The postprandial dysfunction of vascular endothelium was then investigated in an ex vivo model, by a transcriptomic approach. This study reveals that postprandial hyperlipæmic serum has an antiproliferative effect on vascular endothelial cells and leads to genne expression variations in favour of cell cycle arrest and of induction of apoptosis in these cells. These results confirm a deleterious effect of the postprandial hyperlipæmic period on the vascular endothelium, and provide new data on the involved molecular mechanisms. In conclusion, our work provides new data for a better understanding of the biological processes involved in the metabolic stress induced after high-fat consumption leading to vascular dysfunction.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Dual IRE1 RNase functions dictate glioblastoma development

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1 alpha (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.Institut National du Cancer (INCa PLBIO: 2017-148 PLBIO: 2015-111 INCA_ 7981 La Ligue Contre le Cancer (Comite des Landes, LARGE project) PHC Maimonide EU H MSCA ITN-675448 RISE-734749 Region Bretagne "AAP CRITT sante" French government Fondation pour la Recherche Medicale Fondation de France Region Bretagn

Transcriptomic analysis of aorta from a short-term high-fat diet fed mouse reveals changes in the expression of vessel structure genes

International audienceHigh-density microarrays were recently used to identify the genomic profiles of vascular cells during atherogenesis. This strategy succeeded in identifying both biomarkers and underlying biological processes of the pathological development. However, data documenting the early stages of disease are sparse. To identify the mechanisms involved in atherogenesis, we examined differential gene expression in the aorta of C57BL/6J mice fed a high-fat diet (HFD) or a low-fat diet (LFD), for a short period of time of three weeks. The cDNAmicroarray analysis revealed that the expression of 448 genes was significantly different between the two groups. As expected, key genes involved in lipid synthesis or catabolism were down- and upregulated, respectively, representing a normal gene expression response to increased cellular lipid levels. Overrepresented biological processes were identified by Gene Ontology (GO) analysis, which revealed that aortic cells differentiate into a new phenotype in mice fed the HFD. This phenotype was represented by changes in the expression of 81 genes associated with extracellular matrix and cytoskeletal modifications. Some of these genes were previously shown to be involved in the cardiovascular diseases process. In conclusion, short-term HFD consumption results in metabolic disturbances leading to a broad induction of genes involved in vessel architecture remodelling

Intracellular ATP levels determine cell death fate of cancer cells exposed to both standard and redox chemotherapeutic agents.

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined

MicroRNA-1291-mediated silencing of IRE1 enhances Glypican-3 expression

International audienceMicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3' untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5' UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3' UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

Increase in cytosolic calcium concentration ([Ca(2+)](c)), release of endoplasmic reticulum (ER) calcium ([Ca(2+)](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca(2+)](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca(2+)](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca(2+)](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca(2+)](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca(2+)](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells

Novel roles of the unfolded protein response in the control of tumor development and aggressiveness

International audienceThe hallmarks of cancer currently define the molecular mechanisms responsible for conferring specific tumor phenotypes. Recently, these characteristics were also connected to the status of the secretory pathway, thereby linking the functionality of this cellular machinery to the acquisition of cancer cell features. The secretory pathway ensures the biogenesis of proteins that are membrane-bound or secreted into the extracellular milieu and can control its own homeostasis through an adaptive signaling pathway named the Unfolded Protein Response (UPR). In the present review, we discuss the specific features of the UPR in various tumor types and the impact of the selective activation of this pathway on cell transformation, tumor development and aggressiveness

Control of the Unfolded Protein Response in Health and Disease

International audienceThe unfolded protein response (UPR) is an integrated, adaptive biochemical process that is inextricably linked with cell homeostasis and paramount to maintenance of normal physiological function. Prolonged accumulation of improperly folded proteins in the endoplasmic reticulum (ER) leads to stress. This is the driving stimulus behind the UPR. As such, prolonged ER stress can push the UPR past beneficial functions such as reduced protein production and increased folding and clearance to apoptotic signaling. The UPR is thus contributory to the commencement, maintenance, and exacerbation of a multitude of disease states, making it an attractive global target to tackle conditions sorely in need of novel therapeutic intervention. The accumulation of information of screening tools, readily available therapies, and potential pathways to drug development is the cornerstone of informed clinical research and clinical trial design. Here, we review the UPR's involvement in health and disease and, beyond providing an in-depth description of the molecules found to target the three UPR arms, we compile all the tools available to screen for and develop novel therapeutic agents that modulate the UPR with the scope of future disease intervention