370 research outputs found

    Potentiel thérapeutique de l'inhibition d'HDAC6 en hypertension artérielle pulmonaire

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    Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2017-2018L’hypertension artérielle pulmonaire (HTAP) est une maladie vasculaire incurable encore incomplètement comprise. Elle se caractérise cliniquement par une élévation de la pression moyenne dans l’artère pulmonaire (AP) au-delà du seuil de 25 mmHg. Au niveau cellulaire, cette élévation des pressions est attribuable à une prolifération excessive et une résistance à l’apoptose accrue des cellules musculaires lisses (CML) d’AP. HDAC6 est une histone désacétylase 6 principalement cytoplasmique régulant divers mécanismes de survie surexprimée en réponse au stress dans plusieurs cancers. Étant donné les similarités entre les cellules cancéreuses et les cellules vasculaires HTAP, nous avons émis l’hypothèse qu’HDAC6 est surexprimée en HTAP et contribuait au phénotype prolifératif et anti-apoptotique des CMLAPs et au remodelage vasculaire en HTAP. À l’aide de souris génétiquement modifiées et d’approches pharmacologiques dans deux modèles animaux précliniques d’HTAP, nous avons voulu démontrer qu’HDAC6 représente une cible thérapeutique de choix. Nous faisons la démonstration qu’HDAC6 est surexprimée dans les tissus pulmonaires, les AP distales et les CML isolées de patients HTAP lorsque comparés aux donneurs sains. L’inhibition moléculaire et pharmacologique d’HDAC6 réduit la prolifération et la résistance à l’apoptose des CMLAPs HTAP, sans avoir d’effet sur les cellules contrôles. D’un point de vue mécanistique, nous démontrons qu’HDAC6 désacétyle KU70, bloquant la translocation mitochondriale de Bax pour éluder l’apoptose. L’inhibition d’HDAC6 in vivo par la tubastatine A améliore significativement les paramètres hémodynamiques et le remodelage vasculaire dans deux modèles d’HTAP précliniques. Nous montrons que l’inhibition d’HDAC6 peut être combinée de façon sécuritaire à une bithérapie HTAP approuvée présentement utilisée en clinique et que la trithérapie proposée procure un effet bénéfique additif à l’inhibition d’HDAC6 seule sur le remodelage. Finalement, nous montrons que les souris mutées pour HDAC6 ont un remodelage vasculaire et une élévation des pressions artérielles pulmonaires significativement moins grands que les souris non mutées en réponse à une hypoxie de 3 semaines. Nous démontrons pour la première fois l’implication d’HDAC6 dans le développement de l’HTAP. L’inhibition d’HDAC6 semble être une avenue thérapeutique intéressante dans le traitement de l’HTAP. La tubastatine A étant déjà en phase clinique dans le traitement de certains cancers, l’évaluation de son efficacité pour la clientèle HTAP pourrait être rapidement mise en place.RATIONALE: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with limited therapeutic options. Although exposed to stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) exhibit a pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase implicated in the regulation of multiple pro-survival mechanisms and overexpressed in response to stress in cancer cells. Due to the similarities between cancer and PAH, we hypothesized that HDAC6 expression is increased in PAH-PASMCs to face stress, allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH. OBJECTIVE: Using genetically modified mice and pharmacological approaches, we aimed to demonstrate that HDAC6 inhibition is a promising strategy to improve PAH. METHODS AND RESULTS: HDAC6 is significantly up-regulated in lungs, distal PAs and isolated PASMCs from PAH patients and animal models. Molecular and pharmacological inhibition of HDAC6 reduces PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) in vitro sparing control cells. Mechanistically, we demonstrate that HDAC6 deacetylates Ku70, blocking the translocation of Bax to the mitochondria and preventing apoptosis. In vivo inhibition of HDAC6 (Tubastatin A) significantly improves established PAH in two experimental models (Sugen/hypoxia and monocrotaline) and can be safely given in combination with currently approved PAH therapies. Finally, Hdac6 K.O mice have significantly lower right ventricle systolic pressure in response to 3 weeks of chronic-hypoxia compared to wild-type mice. CONCLUSION: We showed for the first time that HDAC6 is implicated in PAH development and represents a new promising therapeutic target to improve PAH

    Role of Long Noncoding RNAs in Diabetic Complications

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    LncRNAs, recently found to be pervasively transcribed in the genome, can play key roles in epigenetic modifications during gene transcription. Their role in diabetic complications are however not clear. Using array analysis, we identified alterations of two lncRNAs in high glucose (25mM/L,HG) treated retinal endothelial cells (HRECs). ANRIL expressions were further measured with or without siRNA knockdown in ECs. ANRIL knockout (KO) mice with or without streptozotocin (STZ)-induced diabetes were also investigated for mRNA and protein expression of VEGF and ECM proteins FN and Col1α4. ANRIL knockdown prevented glucose-induced increased VEGF, FN and Col1α4 levels. Comparable results were observed in the retina, heart and kidneys of diabetic ANRILKO mice compared to wildtype controls. We further showed that these alterations under ANRIL’s regulation are mediated by p300 and enhancer of zeste 2 (EZH2) of the PRC2 complex. We also investigated the role of H19 in diabetes by silencing or overexpressing H19 in ECs exposed to various glucose levels. We extended our study to H19 knockout (KO) mice and vitreous samples from patients with proliferative DR. In both instances, diabetes caused downregulation of H19 expression. H19 overexpression prevented glucose-induced endothelial-mesenchymal transition (EndMT) through TGF-β in a Smad-independent pathway. Additional experiments showed a regulatory relationship between ANRIL and H19. These data suggest that glucose and diabetes cause alteration of specific lncRNAs in ECs and mouse tissues respectively. Identification of such mechanisms help in a better understanding of the pathologies in diabetes and consequent development of RNA based therapies

    Regulating the Regulators: The Post-Translational Code of Class I HDAC1 and HDAC2

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    Class I histone deacetylases (HDACs) are cellular enzymes expressed in many tissues and play crucial roles in differentiation, proliferation, and cancer. HDAC1 and HDAC2 in particular are highly homologous proteins that show redundant or specific roles in different cell types or in response to different stimuli and signaling pathways. The molecular details of this dual regulation are largely unknown. HDAC1 and HDAC2 are not only protein modifiers, but are in turn regulated by post-translational modifications (PTMs): phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and carbonylation. Some of these PTMs occur and crosstalk specifically on HDAC1 or HDAC2, creating a rational “code” for a differential, context-related regulation. The global comprehension of this PTM code is central for dissecting the role of single HDAC1 and HDAC2 in physiology and pathology

    Cardiac Fibrosis in heart failure: Focus on non-invasive diagnosis and emerging therapeutic strategies

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    Heart failure is a leading cause of mortality and hospitalization worldwide. Cardiac fibrosis, resulting from the excessive deposition of collagen fibers, is a common feature across the spectrum of conditions converging in heart failure. Eventually, either reparative or reactive in nature, in the long-term cardiac fibrosis contributes to heart failure development and progression and is associated with poor clinical outcomes. Despite this, specific cardiac antifibrotic therapies are lacking, making cardiac fibrosis an urgent unmet medical need. In this context, a better patient phenotyping is needed to characterize the heterogenous features of cardiac fibrosis to advance toward its personalized management. In this review, we will describe the different phenotypes associated with cardiac fibrosis in heart failure and we will focus on the potential usefulness of imaging techniques and circulating biomarkers for the non-invasive characterization and phenotyping of this condition and for tracking its clinical impact. We will also recapitulate the cardiac antifibrotic effects of existing heart failure and non-heart failure drugs and we will discuss potential strategies under preclinical development targeting the activation of cardiac fibroblasts at different levels, as well as targeting additional extracardiac processes

    MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers

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    Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-dependent nuclear receptors, which control the transcription of genes involved in energy homeostasis and inflammation and cell proliferation/differentiation. Alterations of PPARs’ expression and/or activity are commonly associated with metabolic disorders occurring with obesity, type 2 diabetes, and fatty liver disease, as well as with inflammation and cancer. Emerging evidence now indicates that microRNAs (miRNAs), a family of small noncoding RNAs, which fine-tune gene expression, play a significant role in the pathophysiological mechanisms regulating the expression and activity of PPARs. Herein, the regulation of PPARs by miRNAs is reviewed in the context of metabolic disorders, inflammation, and cancer. The reciprocal control of miRNAs expression by PPARs, as well as the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNA, is also discussed

    Study of myocardial pathological fibrosis using protein-protein-directed nanomaterials as theracnostic tool and targeted gene expressin system

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    RESUMEN La tesis aquí escrita pretende abordar el estudio de la fibrosis cardiaca patológica, con el objetivo de esclarecer y contribuir al desarrollo de nuevas aproximaciones a su tratamiento. Los medios para alcanzar estos objetivos se basan en la utilización de nanomateriales dirigidos por proteínas como herramienta teragnóstica, así como la implementación de un sistema de expresión genética dirigido. Como rasgo distintivo de este estudio, se establecerá un sistema de investigación traslacional, mediante la utilización y evolución de los sistemas utilizados en el trabajo, con el claro objetivo final, de que el trabajo aquí presentado pueda ser utilizado para futuras investigaciones.ABSTRACT This thesis aims to address the study of pathological cardiac fibrosis, with the objective of clarifying and contributing to the development of new approaches to its treatment. The means to achieve these objectives are based on the use of protein targeted nanomaterials as a theragnostic tool, as well as the implementation of a targeted gene expression system. As a distinctive feature of this study, a translational research system will be established through the use and evolution of the systems used in the work, with the clear final objective that the work presented here can be used for future research

    Investigating the role of epigenetics in the regulation of inflammatory skin disease

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    PhD ThesisPsoriasis represents a complex interplay between genetic predisposition, the environment and inflammatory responses, and increasing evidence suggests alterations in the epigenome, including histone acetylation, plays a role. Bromodomain-containing proteins regulate gene expression by binding to acetyl-lysine residues on histones and recruiting transcription factors to gene regulatory regions. The development of small molecule inhibitors of bromodomain extraterminal (BET) proteins has enabled interrogation of this pathway. Interestingly BET inhibitors demonstrate anti-proliferative and anti-inflammatory effects in in vitro and in vivo models of cancer and inflammation. An established in-vitro keratinocyte model of cutaneous inflammation was further developed to characterise IL-6 and IL-8 (mRNA and protein) responses to TNF+ IL-17 stimulation. Chromatin immunoprecipitation (ChIP) studies showed psoriasis-relevant stimuli induced dynamic, gene-specific alterations of the epigenome, including histone hyperacetylation, with co-ordinated recruitment of BET proteins (Brd2, Brd3 and Brd4) and RNA polymerase II, to the promoter region of IL-6 and IL-8. The effects of TNF+ IL-17 stimulation in keratinocytes were validated through global gene expression array studies which showed stimulation modulated expression of keratinocyte genes known to be differentially expressed in psoriasis and involved in its pathogenesis. The hypothesis that BET proteins are involved in regulating inflammatory responses in keratinocytes was tested using a specific BET inhibitor, I-BET151; this blocked pathogenic inflammatory responses. In particular, IL-6 and IL-8 responses to TNF + IL-17 stimulation demonstrated potent sensitivity to I-BET151 treatment; this could be accounted for by the decreased binding of BET proteins and RNA polymerase II to IL-6 and IL-8 gene promoter regions in the presence of the BET inhibitor. Global gene expression array studies showed genes sensitive to BET inhibition were primarily involved in the cell cycle and inflammation, with many relevant to the pathogenesis of psoriasis. In addition, ~20% of genes identified in a previously published meta-analysis of five psoriasis transcriptomic studies were differentially modulated by I-BET151 treatment in TNF + IL-17 stimulated NHEKs. Furthermore, acetate, a principle metabolite of ethanol, a factor implicated in the development and exacerbation of psoriasis, enhanced IL-6, but not IL-8, responses to TNF + IL-17 stimulation through gene-specific epigenetic modifications at the promoter region. IFN also enhanced IL-6, but not IL-8, response to TNF + IL-17 stimulation, suggesting i) IL-6 is more sensitive to enhancement by additional disease relevant stimuli and ii) IL-6 and IL-8 are differentially regulated at the transcriptional level; ChIP studies showed increased enrichment of Brd4/p65 at the IL-6 promoter compared to the IL-8 promoter.The Wellcome Trus

    Oxidative stress in cardiac hypertrophy: From molecular mechanisms to novel therapeutic targets.

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    When faced with increased workload the heart undergoes remodelling, where it increases its muscle mass in an attempt to preserve normal function. This is referred to as cardiac hypertrophy and if sustained, can lead to impaired contractile function. Experimental evidence supports oxidative stress as a critical inducer of both genetic and acquired forms of cardiac hypertrophy, a finding which is reinforced by elevated levels of circulating oxidative stress markers in patients with cardiac hypertrophy. These observations formed the basis for using antioxidants as a therapeutic means to attenuate cardiac hypertrophy and improve clinical outcomes. However, the use of antioxidant therapies in the clinical setting has been associated with inconsistent results, despite antioxidants having been shown to exert protection in several animal models of cardiac hypertrophy. This has forced us to revaluate the mechanisms, both upstream and downstream of oxidative stress, where recent studies demonstrate that apart from conventional mediators of oxidative stress, metabolic disturbances, mitochondrial dysfunction and inflammation as well as dysregulated autophagy and protein homeostasis contribute to disease pathophysiology through mechanisms involving oxidative stress. Importantly, novel therapeutic targets have been identified to counteract oxidative stress and attenuate cardiac hypertrophy but more interestingly, the repurposing of drugs commonly used to treat metabolic disorders, hypertension, peripheral vascular disease, sleep disorders and arthritis have also been shown to improve cardiac function through suppression of oxidative stress. Here, we review the latest literature on these novel mechanisms and intervention strategies with the aim of better understanding the complexities of oxidative stress for more precise targeted therapeutic approaches to prevent cardiac hypertrophy
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