Caractérisation des souches de Pseudomonas aeruginosa isolées des patients atteints de la fibrose kystique par différentes méthodes de typage

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Measures of high-density lipoprotein function in men and women with severe aortic stenosis

Background: Calcification of the aortic valve is a common heart valve disorder, in some cases leading to clinically impactful severe aortic stenosis (AS). Sex-specific differences in aortic valve calcification (ACV) exist, with women having a lower burden of calcification than men as measured by computed tomography; however, the pathophysiological mechanism that leads to these differences remains unclear. Methods: Using cultured human Tamm-Horsfall protein 1 (THP-1) macrophages and human aortic valve interstitial cells, the effects of high-density lipoprotein (HDL) particles isolated from the plasma of men and women with severe AS were studied for cholesterol efflux capacity (CEC). Results: HDL-CEC was assessed in 46 patients with severe AS, n = 30 men, n = 16 women. ATP-Binding Cassette A1 (ABCA1)-mediated HDL-CEC was measured from human cultured THP-1 macrophages to plasma HDL samples. Women with severe AS had more ABCA1-mediated HDL-CEC, as compared to men (8.50 ± 3.90% cpm vs. 6.80 ± 1.50% cpm, P = 0.04). HDL pre-β1 and α-particles were higher in women than in men by spectral density, (pre-β1 HDL, 20298.29 ± 1076.15 vs. 15,661.74 ± 789.00, P = 0.002, and α-HDL, 63006.35 ± 756.81 vs. 50,447.00 ± 546.52, P =0.03). Lecithin-cholesterol acyltransferase conversion of free cholesterol into cholesteryl esters was higher in women than men (16.44 ± 9.11%/h vs. 12.00 ± 8.07%/h, P = 0.03). Conclusions: Sex-specific changes in various parameters of HDL-CEC were found in patients with severe AS. Sex-based modifications in HDL functionality by HDL-CEC might account for the reduced burden of calcification in women vs. men with severe AS. Therefore, future studies should target sex-related pathways in AS to help to improve understanding and treatment of AS

Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

Objective Probucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. Results The inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high-density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. Conclusions We report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated, or in which ORP2 expression is disrupted. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder

Vulnerable Plaque, Characteristics, Detection, and Potential Therapies

Plaque development and rupture are hallmarks of atherosclerotic vascular disease. Despite current therapeutic developments, there is an unmet necessity in the prevention of atherosclerotic vascular disease. It remains a challenge to determine at an early stage if atherosclerotic plaque will become unstable and vulnerable. The arrival of molecular imaging is receiving more attention, considering it allows for a better understanding of the biology of human plaque and vulnerabilities. Various plaque therapies with common goals have been tested in high-risk patients with cardiovascular disease. In this work, the process of plaque instability, along with current technologies for sensing and predicting high-risk plaques, is debated. Updates on potential novel therapeutic approaches are also summarized

HDL biogenesis and function: relevance to atherosclerotic vascular disease prevention and novel therapies

Atherosclerosis is the most common cause of myocardial infarctions and stroke in Canada and the world. Strong epidemiological evidence exists to support high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for coronary artery disease (CAD). However, this paradigm is not supported by Mandelian randomization. Complexity in HDL functions makes the development of a single measurement challenging. We believe that a better understanding of HDL antiatherogenic functions as a therapeutic target including its important function into reverse cholesterol transport, will allow novel insight on novel emergent therapies of modulating HDL. This thesis consists of five inter-related studies with the central unifying theme of HDL physiology. First, in three reviews (of which two were as invited author); we discussed the controversy of HDL-C raising therapies including torcetrapib, dalcetrapib, evacetrapib and niacin. We highlight the paradigm based on HDL function to understand the current clinical equipoise and the neutrality of clinical benefit of HDL-C raising therapies. We emphasize that HDL-C is an imperfect metric of HDL functionality and thus inadequate predictor of disease risk. Second, we develop an HDL mediated cholesterol efflux assay to study HDL function and biogenesis. We optimized the standardization of this assay and demonstrate how fine-tuning the experimental protocol streamlines measurements and avoid inconsistencies in comparing data. Third, we evaluated two novel 26-amino acid apolipoprotein mimetic peptide (ATI-5261) and (CS-6253) designed from the carboxyl terminal sequence of apoE, in their ability to mimic apoA-I functionality to RCT in vitro and ex vivo. This study suggests that peptides mimic apo A-I in the process of HDL biogenesis. Fourth, we confirmed the formation of ATP-binding cassette transporter-1 (ABCA1)-dependent microparticles (MPs) during HDL biogenesis from various cell lines when incubated with apo A-I or alone, and that theses MPs transport cellular cholesterol. Fifth, in a translational study we examine the HDL cholesterol efflux capacity in acute coronary syndromes (ACS) and carotid atherosclerosis patients. We show that HDL mediated cholesterol efflux is associated with atherosclerotic burden as expressed by carotid intima-media thickness and the prevalence rate of CAD independently of HDL-C levels.In summary, this work contributed to the preclinical development of novel apoA-I mimetic compounds that modulate HDL function. It has brought to light novel assay for measuring HDL cholesterol efflux. We believe that HDL function and biogenesis together are of great importance to develop compound targeting HDL levels. However, it has to be determined if improved HDL function will reduce residual CVD risk in treated patients where HDL-C based therapies apparently failed to prevent. Finally, there is a considerable interest in harnessing the power of HDL to reverse atherosclerosis and prevent or treat vascular events.L'athérosclérose est la cause la plus fréquente d'accidents vasculaires cérébraux ou l'infarctus du myocarde au Canada et dans le monde. De fortes évidences épidémiologiques supportent le cholestérol lie aux lipoprotéines de haute densité (C-HDL) comme facteur de risque indépendant de la maladie coronarienne athéroscléreuse (MCAS). Cependant, ce paradigme n'est pas supporté par les études de randomisation Mendéliennes. La complexité des fonctions des HDL rend pour le moins très difficile le développement d'un test de mesure. Nous croyons qu'une meilleure compréhension des fonctions anti-athérogènes des particules HDL comme cible thérapeutique, y compris leur rôle dans le transport inverse du cholestérol, permettra d'avoir un nouvel aperçu sur de nouveaux traitements des maladies cardiovasculaires. Cette thèse se compose de cinq études liées entre eux avec le thème central concentrée sur la physiologie des particules HDL. Tout d'abord, dans trois revues dont deux comme ''auteur invité'', nous avons la controverse des thérapies basées sur le niveau du C-HDL (torcetrapib, dalcetrapib, evacetrapib et niacine). Nous avons mis en évidence le paradigme basé sur la fonction des HDL pour comprendre l'équilibre clinique actuel et la neutralité du bénéfice clinique des thérapies visant à élever le C-HDL. Nous avons montré que la mesure du C-HDL est une mesure imparfaite de la fonctionnalité des HDL et donc un prédicteur inadéquat du risque. Deuxièmement, nous avons développé un test d'efflux de cholestérol pour étudier la biogénèse et fonction des HDL. Nous avons établi plus de standardisation à ce test et démontré comment optimiser des divers écueils dans le protocole expérimental afin de rationaliser toutes les mesures et d'éviter toute incompatibilité dans la comparaison des données. En troisième lieu, nous avons évalué deux nouveaux peptides de 26-acides aminés (ATI-5261) et (CS-6253) conçu à partir de la séquence carboxyle-terminale de l'apoE, dans leur capacité à imiter le rôle de l'apolipoprotein A-I (apoA-I) dans le transport inverse de cholesterol in vitro et ex vivo. Quatrièmement, nous avons confirmé le rôle du transporteur ABCA1 dans la formation de microparticules (MPs) au cours de la biogenèse des HDL dans diverses cellules. Ces MPs transportent du cholesterol. Cinquièmement, dans une étude transrationnelle, nous avons examiné la capacité des HDL dans l'efflux de cholesterol dans le syndrome coronarien aigue et les malades avec l'athérosclérose carotidienne. Nous avons montré que la fonction HDL dans l'efflux de cholesterol est inversement associée à la sténose carotidienne tel qu'exprimées par l'épaisseur de l'intima et à la maladie cardiovasculaire indépendamment de C-HDL.En résumé, ce travail a contribué au développement préclinique de nouveaux apoA-I mimétiques qui modulent la fonction des HDL. Il a mis en lumière de nouvelles méthodes régissant l'efflux de cholestérol par les HDL. Nous croyons que le rôle et la biogenèse des HDL sont des cibles thérapeutiques de grande importance pour développer et tester des nouvelles thérapies. Cependant, il reste à déterminer si la fonction améliorée des HDL réduira le risque résiduel de MCAS là où les thérapies basées sur le C-HDL n'ont pas parvenues pas à empêcher. Enfin, cela permet de mieux évaluer le potentiel thérapeutique permettant de moduler la fonction HDL dans la prévention ou le traitement d'athérosclérose. Enfin, il y a un intérêt considérable en exploitant la puissance des particules HDL pour inverser l'athérosclérose et de prévenir ou de traiter des événements vasculaires

Measures of High-Density Lipoprotein Function and Composition in Men and Women with Severe Aortic Valve Stenosis

Introduction and Hypothesis: Aortic valve calcification (AVC), is the intrinsic mechanism of valvular obstruction in severe aortic stenosis (AS), sharing similarities with atherosclerosis. There are differences in AVC by sex, with women having a lower burden of calcification than men, but the mechanism for this is unclear. Improved HDL functionality has been associated with protection against coronary artery calcification, but its relationship to AVC is less studied.Methods: We evaluated measures of high-density lipoprotein (HDL) functionality in 46 patients with severe AS, n= 30 men, n=16 women. ATP-Binding Cassette A1 (ABCA1) mediated cholesterol efflux capacity (CEC) was measured from human cultured Tamm-Horsfall protein 1 (THP-1) macrophages to apoB-depleted patient plasma samples. Lipid profile, lecithin-cholesterol acyltransferase activity (LCAT), fast protein liquid chromatography (FPLC), HDL size, and small pre-β1 and α-HDL particles, were measured in apolipoprotein B-depleted patient plasma. Proteomic analysis was used to explore sex-based differences in the HDL proteome.Results: Women with severe AS had higher ABCA1-mediated CEC, as compared to men (n=46, p=0.04). We found differences in FPLC profiles between sexes in severe AS along with a reduction in preβ and α-particles in men vs women by spectral counts, (preβ-HDL, 15661.74±789.00 vs 20298.29±1076.15, p=0.002), and (α-HDL, 50447.00± 546.52 vs 63006.35±756.81, p=0.03). Conversion of free cholesterol into cholesteryl esters by LCAT was decreased in men vs. women (12.00±8.07 %/h vs 16.44±9.11 %/h, p=0.03). We identified 108 HDL-associated proteins in a representative man and woman, of which, 15 were found only in the female sampleConclusions: Sex-specific differences in measures of HDL functionality were found in patients with severe AS. Future studies should consider whether sex-based differences in HDL functionality might account for the decreased burden of calcification in women vs. men with severe AS

Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic