11 research outputs found

    Implication d'EZH2 en hypertension artérielle pulmonaire

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    L'hypertension artérielle pulmonaire (HTAP), caractérisée par une pression artérielle pulmonaire moyenne (mPAP) supérieure à 20mmHg au repos, est une pathologie progressive et létale. L'augmentation progressive de la pression artérielle pulmonaire est la résultante d'une vasoconstriction soutenue associée à un profond remodelage des artères pulmonaires distales. Le remodelage vasculaire est dû principalement à une prolifération anormalement élevée et une résistance importante à l'apoptose des cellules musculaires lisses des artères pulmonaires (CMLAPs). Le remodelage vasculaire et la vasoconstriction engendre une obstruction progressive des artères pulmonaires favorisant une augmentation des résistances vasculaires pulmonaires. Cette augmentation progressive des résistances vasculaires pulmonaires se répercute sur le ventricule droit (VD) forçant celui-ci à s'hypertrophier pour compenser et maintenir ses fonctions. Néanmoins, à mesure que la maladie progresse, l'obstruction artérielle s'accentue, rendant la compensation cardiaque droite plus difficile ce qui mènera ultimement au décès des patients par défaillance cardiaque droite. À ce jour les outils thérapeutiques utilisés ciblent uniquement le tonus vasculaire en favorisant la vasodilatation. Cependant, cette stratégie thérapeutique n'améliore que de façon médiocre le taux de survie des patients ce qui rend urgent le besoin de développer de nouvelles stratégies ciblant l'obstruction artérielle pulmonaire. Le remodelage, étant la conséquence d'une prolifération et d'une survie cellulaire importante, met en exergue une certaine similitude avec le cancer. En effet, certaines voies cellulaires dérégulées dans le cancer, comme ici la prolifération ou la survie cellulaire, se retrouvent également dérégulées en hypertension artérielle pulmonaire. La littérature scientifique démontre de plus en plus l'implication de modifications épigénétiques dans le développement de cancer, notamment par la transcription de gènes pro-prolifératifs et anti-apoptotiques. Les dernières années de recherche ont démontré que des facteurs épigénétiques pouvaient être responsables du développement et de la progression de l'HTAP. En ce sens, nous nous sommes intéressés au facteur épigénétique EZH2 (Enhancer of Zeste Homologue 2) fortement impliqué dans la prolifération et la survie cellulaire dans de nombreux cancers, faisant de lui une cible préférentielle des thérapies anticancéreuses. Étant donné le phénotype pro-prolifératif et résistant des CMLAPs-HTAP, EZH2 constitue une cible à étudier dans le remodelage vasculaire. De plus, la littérature rapporte un effet cardioprotecteur d'EZH2 face à l'hypertrophie et la fibrose dans le tissu cardiaque. Bien que l'HTAP soit une pathologie vasculaire avant tout, les patients décèdent d'une défaillance cardiaque droite. Le ventricule droit n'étant plus apte à s'adapter, accumule de la fibrose rendant la contraction laborieuse ce qui mène à la défaillance cardiaque droite. À la vue de son rôle de protecteur cardiaque décrit dans la littérature, nous avons également étudié sa potentielle implication dans l'hypertrophie et la fibrose cardiaque HTAP. Dans un premier volet de l'étude, nous avons démontré qu'EZH2 est surexprimé dans les cellules musculaires lisses des artères pulmonaires de patients HTAP et de modèles expérimentaux. In vitro, l'inhibition d'EZH2 diminue la prolifération et la résistance à l'apoptose des cellules musculaires lisses HTAP comparativement aux cellules contrôles. Par une approche multiomique, nous avons démontré que l'inhibition d'EZH2 diminue l'expression de nombreux facteurs impliqués dans la progression du cycle cellulaire, y compris les cibles E2F, ainsi que des protéines impliquées dans la fonction mitochondriale. De plus, nous avons également démontré que l'inhibition d'EZH2 altère la bioénergétique des CMLAPs-HTAP. Dans le second volet de notre étude, nous avons démontré qu'EZH2 se retrouve surexprimé dans les ventricules droits compensés des patients et des modèles expérimentaux. Nous avons démontré qu'une inhibition de l'expression d'EZH2, in vitro, favorise l'hypertrophie cellulaire. D'un point de vue mécanisme cellulaire, EZH2 est régulé par l'ARN long non codant H19, plus précisément son micro-ARN : miR-675. Nous avons démontré que cette régulation passe par l'intermédiaire du facteur de transcription E2F1 qui régulé par miR-675 va réguler à son tour l'expression d'EZH2. L'inhibition d'H19 in vivo induit la surexpression d'E2F1 et d'EZH2 et démontre un effet thérapeutique cardioprotecteur bénéfique dans deux modèles expérimentaux de défaillance cardiaque droite. Pour conclure, les deux volets de notre étude nous permettent de mettre en évidence une implication d'EZH2 dans le phénotype pro-prolifératif et anti-apoptotique des CMLAPs-HTAP et également un rôle protecteur de la fonction ventriculaire droite. Ainsi, cela ouvre de nouvelles possibilités de découvertes thérapeutiques.Pulmonary arterial hypertension (PAH), characterized by a mean pulmonary arterial pressure (mPAP) greater than 20mmHg at rest, is a progressive and fatal condition. The progressive increase in pulmonary artery pressure is the result of sustained vasoconstriction associated with a deep remodeling of the distal pulmonary arteries. Vascular remodeling is mainly due to abnormal proliferation and increased survival of pulmonary artery smooth muscle cells (PASMCs). Vascular remodeling and vasoconstriction cause progressive obstruction of the pulmonary arteries, promoting an increase in pulmonary vascular resistance. This progressive increase in pulmonary vascular resistance affects the right ventricle (RV) forcing it to compensate by hypertrophy to maintain its functions. However, as the disease progresses, the arterial obstruction worsens, making right cardiac compensation more difficult, which will ultimately lead to the death of patients from right heart failure. To date, the therapeutic tools used only target vascular tone by promoting vasodilation. However, this therapeutic strategy poorly improves patient survival rates, which makes the need to develop new strategies targeting pulmonary artery obstruction urgent. The remodeling being the consequence of a high proliferation and an important cell survival highlights some similarities with cancer. Indeed, some deregulated cell pathways in cancer, such as cell proliferation or survival, are also deregulated in pulmonary arterial hypertension. The scientific literature increasingly demonstrates the involvement of epigenetic modifications in the development of cancer, through the transcription of pro-proliferative and anti-apoptotic genes. The last years of research have shown that epigenetic factors could be responsible for the development and progression of the PAH. In this sense, we were interested in the epigenetic factor EZH2 (Enhancer of Zeste Homologue 2) strongly involved in cell proliferation and survival in many cancers, making it a preferential target for anticancer therapies. Given the hyper-proliferative and resistant phenotype of PAH-PASMCs, EZH2 constitutes a target to be studied in vascular remodeling. In addition, the literature reports a cardioprotective effect of EZH2 against hypertrophy and fibrosis in heart tissue. Although PAH is primarily a vascular disease, patients die of right heart failure. As the right ventricle is no longer able to adapt, fibrosis accumulates making ventricular contractility difficult which leads to right heart failure. In view of its role as a cardiac protector described in the literature, we investigated its potential involvement in cardiac hypertrophy and fibrosis in PAH. In a first part of our study, we demonstrated that EZH2 is overexpressed in PAH-PASMCs from patients and in experimental models. In vitro, EZH2 inhibition decreases the proliferation and resistance to apoptosis of PAH-PASMCs compared to control cells. A multi-omic approach has made it possible to highlight multiple targets of EZH2, like targets of the transcription factor E2F1, involved in the regulation of the cell cycle, as well as proteins involved in mitochondrial function. In addition, we have also shown that inhibition of EZH2 alters PAH-PASMCs bioenergetics. In a second part of our study, we demonstrated that EZH2 is overexpressed in compensated right ventricles in PAH patients and in experimental models. We have shown that inhibition of EZH2 expression in vitro promotes cell hypertrophy. From a cellular mechanism point of view, EZH2 is regulated by the long non-coding RNA H19, more precisely its microRNA: miR-675. We have demonstrated that this regulation is mediated by the transcription factor E2F1 which, regulated by miR-675, will in turn regulate the expression of EZH2. Inhibition of H19 in vivo induces E2F1 and EZH2 overexpression and demonstrates a beneficial cardioprotective therapeutic effect in two experimental models of right heart failure. In conclusion, the two parts of our study allow us to demonstrate an involvement of EZH2 in the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs and a protective role of right ventricular function. Thus, these results open new possibilities for therapeutic discoveries

    Substitutions of the S4DIV R2 residue (R1451) in NaV1.4 lead to complex forms of paramyotonia congenita and periodic paralyses

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    Abstract Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP). The R1451C substitution was found in one individual with a single attack of hypoPP induced by glucocorticoids. To elucidate the biophysical mechanism underlying the phenotypes, we used the patch-clamp technique to study tsA201 cells expressing WT or R1451C/L channels. Our results showed that both substitutions shifted the inactivation to hyperpolarized potentials, slowed the kinetics of inactivation, slowed the recovery from slow inactivation and reduced the current density. Cooling further enhanced these abnormalities. Homology modeling revealed a disruption of hydrogen bonds in the voltage sensor domain caused by R1451C/L. We concluded that the altered biophysical properties of R1451C/L well account for the PMC-hyperPP cluster and that additional factors likely play a critical role in the inter-individual differences of clinical expression resulting from R1451C/L

    Implication of EZH2 in the Pro-Proliferative and Apoptosis-Resistant Phenotype of Pulmonary Artery Smooth Muscle Cells in PAH: A Transcriptomic and Proteomic Approach

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    Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling

    Multicenter preclinical validation of BET inhibition for the treatment of pulmonary arterial hypertension

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    Rationale: Pulmonary arterial hypertension (PAH) is a degenerative arteriopathy that leads to right ventricular (RV) failure. BRD4 (bromodomain-containing protein 4), a member of the BET (bromodomain and extra-terminal motif) family, has been identified as a critical epigenetic driver for cardiovascular diseases. Objectives: To explore the therapeutic potential inPAHof RVX208, a clinically available BET inhibitor. Methods: Microvascular endothelial cells, smooth muscle cells isolated fromdistal pulmonary arteries of patientswith PAH, rats with Sugen54161hypoxia- ormonocrotaline1shunt-induced PAH, and rats with RV pressure overload induced by pulmonary artery banding were treated with RVX208 in three independent laboratories. Measurements and Main Results: BRD4 is upregulated in the remodeled pulmonary vasculature of patients with PAH, where it regulates FoxM1 and PLK1, proteins implicated in the DNA damage response. RVX208 normalized the hyperproliferative, apoptosisresistant, and inflammatory phenotype of microvascular endothelial cells and smooth muscle cells isolated from patients with PAH. Oral treatment with RVX208 reversed vascular remodeling and improved pulmonary hemodynamics in two independent trials in Sugen54161hypoxia-PAH and in monocrotaline1shunt-PAH. RVX208 could be combined safely with contemporaryPAHstandard of care. RVX208 treatment also supported the pressure-loaded RV in pulmonary artery banding rats. Conclusions: RVX208, a clinically available BET inhibitor, modulates proproliferative, prosurvival, and proinflammatory pathways, potentially through interactions with FoxM1 and PLK1. This reversed the PAH phenotype in isolated PAH microvascular endothelial cells and smooth muscle cells in vitro, and in diversePAH rat models. RVX208 also supported the pressure-loaded RV in vivo. Together, these data support the establishment of a clinical trial with RVX208 in patients with PAH

    Multicenter Preclinical Validation of BET Inhibition for the Treatment of Pulmonary Arterial Hypertension

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    Rationale: Pulmonary arterial hypertension (PAH) is a degenerative arteriopathy that leads to right ventricular (RV) failure. BRD4 (bromodomain-containing protein 4), a member of the BET (bromodomain and extra-terminal motif) family, has been identified as a critical epigenetic driver for cardiovascular diseases. Objectives: To explore the therapeutic potential in PAH of RVX208, a clinically available BET inhibitor. Methods: Microvascular endothelial cells, smooth muscle cells isolated from distal pulmonary arteries of patients with PAH, rats with Sugen5416 + hypoxia- or monocrotaline + shunt-induced PAH, and rats with RV pressure overload induced by pulmonary artery banding were treated with RVX208 in three independent laboratories. Measurements and Main Results: BRD4 is upregulated in the remodeled pulmonary vasculature of patients with PAH, where it regulates FoxM1 and PLK1, proteins implicated in the DNA damage response. RVX208 normalized the hyperproliferative, apoptosis-resistant, and inflammatory phenotype of microvascular endothelial cells and smooth muscle cells isolated from patients with PAH. Oral treatment with RVX208 reversed vascular remodeling and improved pulmonary hemodynamics in two independent trials in Sugen5416 + hypoxia-PAH and in monocrotaline + shunt-PAH. RVX208 could be combined safely with contemporary PAH standard of care. RVX208 treatment also supported the pressure-loaded RV in pulmonary artery banding rats. Conclusions: RVX208, a clinically available BET inhibitor, modulates proproliferative, prosurvival, and proinflammatory pathways, potentially through interactions with FoxM1 and PLK1. This reversed the PAH phenotype in isolated PAH microvascular endothelial cells and smooth muscle cells in vitro, and in diverse PAH rat models. RVX208 also supported the pressure-loaded RV in vivo. Together, these data support the establishment of a clinical trial with RVX208 in patients with PAH
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