The mechanisms of action of the apolipoprotein A-V in the lipid metabolism

Des dyslipidémies tels que l'hypertriglycéridemie et l'hypercholestérolémie constituent des facteurs de risque indépendants dans le développement des maladies cardiovasculaires. Le gène APOA5 récemment identifié, est localisé chez l'homme sur le chromosome 11q23 qui fait partie du cluster APOA1/C3/A4 et il est associé avec des taux de triglycérides plasmatiques très élevés. L'APOA5 humaine est constitué de 4 exons codant pour une protéine de 366 acides aminés l'apolipoprotéine ApoA-V exprimée uniquement dans le foie et associée dans le plasma aux HDL et VLDL. Une diminution importante du taux de triglycérides a était mise en évidence chez les souris sur-exprimant l'APOA5 humaine. Dans la première partie de la thèse nous avons étudié le mécanisme à travers lequel le gène APOA5 humaine induit un effet hypotriglycéridemique. Initialement nous avons trouvé une forte diminution de la concentration de triglycérides chez les souris transgénique APOA5 humaine par rapport aux souris contrôles sauvages, résultat corrélé avec une baisse de l'activité de la LPL dans le plasma post-hépariné. De plus nous avons démontré que l'apoA-V recombinante interagie physiquement in vitro avec la lipoprotéine lipase et lui diminue de manière significative l'activité enzymatique. La baisse de la concentration en apoB et apoC-III obtenue chez les souris transgéniques APOA5 humaine indique une diminution du nombre des particules des VLDL. Pour approfondir le mécanisme de l'APOA5 humaine dans un environnement hyperlipidemique, nous avons croisé les souris transgéniques APOA5 humaine et APOC3 humaine. Nous avons trouvé une diminution du taux de cholestérol et des triglycérides dans VLDL ainsi que des concentrations de apoB et apoC-III. Ces résultats démontrent que l'apoA-V induit une diminution de la taille des VLDL par l'activation de la lipolyse et augmente la clairance des VLDL. En état postprandial, le taux de triglycérides chez les souris transgéniques APOA5 humaine est nettement inférieur par rapport aux souris sauvages. De plus, nous avons démontré qu'en réponse à ce fat load chez les souris APOA5xAPOC3 humaine, l'apoA-V a était redistribué dès HDL primaires vers les VLDL, mais pas l'apoC-III. Cette délocalisation de l'apoA-V vers les VLDL semble limiter l'augmentation de la concentration de triglycérides pas l'activation de la lipoprotéine lipase. En conclusion, l'apoA-V accélère le catabolisme des lipoprotéines riches en triglycérides par l'activation directe de leur hydrolyse par la lipoprotéine lipase et leur élimination du plasma. Des nombreux travaux ont montré que les polymorphismes du gène APOA5 sont associés avec une diminution des triglycérides, un facteur de risque dans le syndrome métabolique. Dans la deuxième partie de la thèse nous recherches ont visé l'association entre les variantes du gène APOA5 et la fréquence du syndrome métabolique dans une population Roumaines urbaine. Nous avons étudié deux polymorphismes de l'APOA5 (-1,131T>C et c.56C>G) et les taux de l'apoA-V chez 279 sujets et nous les avons corrélées avec des paramètres biochimiques plasmatiques. Les sujets ont été partagés en trois groups : contrôle, syndrome métabolique et maladies cardiovasculaires, une deuxième classification prenant en compte leurs poids (corpulence normale, surpoids, obésité modérée et obésité sévère). Nos résultats ont montré que chez les patients présentant le syndrome métabolique avec l'allèle -1,131C on retrouve plus souvent des indices de masse corporelle élevés, des taux de triglycérides plus élevés mais des concentrations de HDL-C diminuées par rapport aux porteurs du gène native. Les patients avec le syndrome métabolique, porteurs de l'allèle c.56G ont le plus souvent des taux de triglycérides élevés et de HDL-C basses, mais ils ne sont pas associés à l'obésité. Les patients avec des maladies cardiovasculaires porteurs des deux variantes du gène APOA5 ont des taux de triglycérides et de glucose plus élevés mais la concentration de HDL-C diminue. Chez les patients obèses nous avons observé un transport d'apoA-V dès HDL vers les VLDL. Dans le groupe des patients avec le syndrome métabolique nous avons mis en évidence une corrélation positive entre les concentrations d'apoA-V totale et HDL-apoA-V avec le taux de triglycérides plasmatiques pour les génotypes -1,131CC et c.56GG et nous avons trouvé une corrélation inverse avec le HDL-C pour le génotype c.56GG. Nos résultats montrent une corrélation entre la présence de l'allèle -1,131C et la prédisposition pour le syndrome métabolique.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells

Clinical data implicate fluctuations of high levels of plasma glucose in cardiovascular diseases. Endothelial cells (EC) are the first cells of the vessel wall exposed to them. Our aim was to evaluate the effects of oscillating glucose (OG) on EC function and to decipher new molecular mechanisms involved. Cultured human ECs (EA.hy926 line and primary cells) were exposed to OG (5/25 mM alternatively at 3 h), constant HG (25 mM) or physiological concentration (5 mM, NG) for 72 h. Markers of inflammation (Ninj-1, MCP-1, RAGE, TNFR1, NF-kB, and p38 MAPK), oxidative stress (ROS, VPO1, and HO-1), and transendothelial transport proteins (SR-BI, caveolin-1, and VAMP-3) were assessed. Inhibitors of ROS (NAC), NF-kB (Bay 11-7085), and Ninj-1 silencing were used to identify the mechanisms of OG-induced EC dysfunction. The results revealed that OG determined an increased expression of Ninj-1, MCP-1, RAGE, TNFR1, SR-B1, and VAMP-3 andstimulated monocyte adhesion. All of these effects were induced bymechanisms involving ROS production or NF-kB activation. NINJ-1 silencing inhibited the upregulation of caveolin-1 and VAMP-3 induced by OG in EC. In conclusion, OG induces increased inflammatory stress, ROS production, and NF-kB activation and stimulates transendothelial transport. To this end, we propose a novel mechanism linking Ninj-1 up-regulation to increased expression of transendothelial transport proteins

CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were measured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC

Aggregated LDL turn human macrophages into foam cells and induce mitochondrial dysfunction without triggering oxidative or endoplasmic reticulum stress.

Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress

Quantifying brain tumor tissue abundance in HR-MAS spectra using non-negative blind source separation techniques

Given high-resolution magic angle spinning (HR-MAS) spectra from several glial tumor subjects, our goal is to differentiate between tumor tissue types by separating the different sources that contribute to the profile of each spectrum. Blind source separation techniques are applied for obtaining characteristic profiles for necrosis, highly cellular tumor and border tumor tissue and providing the contribution (abundance) of each of these tumor tissue types to the profile of each spectrum. The problem is formulated as a non-negative source separation problem. Non-negative matrix factorization, convex analysis of non-negative sources and non-negative independent component analysis methods are considered. The results are in agreement with the pathology obtained by the histopathological examination that succeeded the HR-MAS measurements. Furthermore, an analysis to verify to which extent the dimension of the input space, the input features and the number of sources to be extracted from the HR-MAS data could influence the performance of the source separation is presented. © 2012 John Wiley & Sons, Ltd.status: publishe

Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence

Mitochondrial DNA Together with miR-142-3p in Plasma Can Predict Unfavorable Outcomes in Patients after Acute Myocardial Infarction

Myocardial infarction is one of the leading causes of death worldwide, despite numerous efforts to find efficient prognostic biomarkers and treatment targets. In the present study, we aimed to assess the potential of six microRNAs known to be involved in cardiovascular diseases, cell-free DNA (cfDNA), and mitochondrial DNA (mtDNA) circulating in plasma to be used as prognostic tools for the occurrence of unfavorable outcomes such as major adverse cardiovascular events (MACE) after acute ST-segment elevation myocardial infarction (STEMI). Fifty STEMI patients were enrolled and monitored for 6 months for the occurrence of MACE. Plasma was collected at three time points: upon admission to hospital (T0), at discharge from hospital (T1), and 6 months post-STEMI (T6). Plasma levels of miR-223-3p, miR-142-3p, miR-155-5p, miR-486-5p, miR-125a-5p, and miR-146a-5p, as well as of cfDNA and mtDNA, were measured by RT-qPCR. Results showed that the levels of all measured miRNAs, as well as of cfDNA and mtDNA, were the most increased at T1, compared to the other two time points. In the plasma of STEMI patients with MACE compared to those without MACE, we determined increased levels of miRNAs, cfDNA, and mtDNA at T1. Hence, we used the levels of all measured parameters at T1 for further statistical analysis. Statistical analysis demonstrated that all six miRNAs and cfDNA plus mtDNA levels, respectively, were associated with MACE. The minimal statistical model that could predict MACE in STEMI patients was the combination of mtDNA and miR-142-3p levels, as evidenced by ROC analysis (AUC = 0.97, p < 0.001). In conclusion, the increased plasma levels of mtDNA, along with miR-142-3p, could be used to predict unfavorable outcomes in STEMI patients