L'hypertension pulmonaire : au delà du poumon : signature épigénétique et métabolique des atteintes systémiques en hypertension pulmonaire

L'hypertension pulmonaire (HP) est définie comme un état pathologique de l’élévation des pressions pulmonaires. L’hypertension artérielle pulmonaire (HTAP), à laquelle se rattachent les premiers travaux de doctorat, est une vasculopathie obstructive caractérisée par une oblitération de la lumière des artères pulmonaires distales, conduisant à une augmentation des pressions pulmonaires (PAP), aboutissant à une dysfonction du ventricule droit (VD) qui est la cause majeure de mort de la pathologie. Cette pathologie, longtemps considérée comme purement pulmonaire, est maintenant associée à de nombreuses atteintes systémiques notamment au niveau du VD et du muscle squelettique expliquant l’impact restreint de la thérapeutique actuelle sur la survie et la tolérance à l’effort qui est le symptôme premier des patients HTAP. Par une étude translationelle basée sur l’utilisation d’échantillons humains, nous montrons au cours des chapitres II et III, la présence d’une atteinte systémique de l’angiogenèse, caractérisée par une diminution de la densité capillaire (immunofluorescence) au niveau des muscles squelettiques et des VD décompensés des patients HTAP. Ensuite, par une approche in vitro, menée à partir de culture primaire de cellules endothéliales (CE) extraites de quadriceps et de VD HTAP, nous démontrons la présence d’une dysfonction endothéliale, caractérisée par une altération du potentiel angiogénique (mesurée par test de matrigel) des CE de patients HTAP. Nous identifions ensuite, par immunoblot et q-PCR, le rôle central de l’axe miR126/SPRED1/ERK dans l’altération de l’angiogenèse décrite chez les patients HTAP. En accord avec ces observations, nous montrons que la sur-expression de miR-126 dans les CE malades est capable de rétablir un potentiel angiogénique sain. Finalement, par une approche in vivo, nous montrons que la modulation de miR-126 dans le muscle squelettique et le ventricule droit d’un modèle animal d’HTAP offre, via l’angiogenèse, une nouvelle approche thérapeutique dans l’amélioration de la tolérance à l’effort (expérience de tapis roulant) et de la fonction cardiaque (cathétérisme cardiaque droit en thorax fermé). Au cours des derniers travaux de ma thèse, présentés dans le chapitre IV de ce manuscrit, nous nous sommes intéressés à l’HP consécutive à une pathologie cardiaque gauche dite de groupe 2. Ce groupe représente la forme d’HP la plus fréquente qui, paradoxalement et par manque de modèle préclinique, est également l’une des moins étudiées. De façon intéressante, de récentes études épidémiologiques montrent une forte interconnexion entre le syndrome métabolique (SM) et l'insuffisance cardiaque avec l'éjection préservée (HFpEF), qui deux problèmes majeurs de santé publique dans les pays occidentaux, et l’HP de groupe 2. Au cours de mes travaux, nous avons, dans un premier temps, crée un nouveau modèle animal qui associe dysfonction diastolique (constriction de l’aorte) et SM (nourriture riche en gras, olanzapine), conduisant à l’établissement d’une HP de groupe 2 (cathétérisme cardiaque droit en thorax fermé) avec un remodelage vasculaire pulmonaire accru. Nous montrons ensuite que, chez ces animaux, l’HP est associée à une accumulation de gras viscéral et péricardique, une augmentation du taux sérique d’adipokines, ainsi qu’une augmentation de l’inflammation pulmonaire. Fonctionnellement, nous montrons que l’augmentation de la sécrétion leptine par le gras des animaux HP se traduit par l’activation de STAT3 (Phospho-STAT3) et une augmentation de la prolifération des cellules musculaires lisses des rats HP. Par une approche in vitro, nous montrons que l’activation de STAT3 induite par la leptine est associée, au niveau des cellules musculaires lisses, à une augmentation de la prolifération. Nous démontrons également que l’activation des macrophages induite par la leptine se traduit par une sécrétion accrue de cytokines inflammatoires. Finalement, nous démontrons que l’axe leptine/STAT3 joue un rôle majeur dans l’établissement et la progression de l’HP asssociée à l’HFpEF et au MS, en favorisant, notamment, la prolifération cellulaire et l’inflammation pulmonaire. Pour conclure, l’ensemble de ces travaux ont eu pour ambition de s’éloigner de l’étiologie classique de l’HP pour introduire les atteintes systémiques comme des altérations à part entière et des acteurs majeurs de l’établissement et la progression de la pathologie, qui se doivent d’être pris en compte par les thérapeutiques de demain.Pulmonary hypertension (PH) is a pathological condition defined by an elevation of pulmonary pressures. Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by obliteration of the lumen of the distal pulmonary arteries, leading to an increase of pulmonary arterial pressures (PAP), resulting in right ventricle (RV) failure, which is the major cause of death of the pathology. For a long time, this pathology was exclusively considered as a lung disease, but recent evidences clearly identified systemic impairments, notably in RV and skeletal muscle, in the disease. Interestingly, these observations might explain the limited impact of current therapy on survival and tolerance to exercise, which is the primary symptom Of PAH patients. Using a translational study based on the use of human samples, we show in Chapters II and III the presence of a systemic impairment of angiogenesis, characterized by a decrease in the capillary density (immunofluorescence), in skeletal muscles and decompensated RV of PAH patients. Then, using endothelial cells (EC) freshly extracted from quadriceps and RV of PAH patients we identified an endothelial dysfunction of PAH EC characterized by an impairment of agiogenic potential (measured in vitro by Matrigel assay). Next, using western blot (WB) and q-PCR we identified a key role of miR126 / SPRED1 / ERK axis in the alteration of angiogenesis described in skeletal muscle and RV of PAH patients. Consistent with these observations, we show that over-expression of miR-126 improves angiogenic potential of PAH EC. Finally, using an in vivo approach, we display that artificial modulation of miR-126 in skeletal muscle and RV of PAH animals, increases capillary density and improves exercise tolerance (treadmill experience) and cardiac function (right cardiac catheterization in closed chest). Taken together, these results suggest that miR-126 could represent a new therapeutic avenue in PAH. In chapter IV of this manuscript we were interested in PH due to left heart disease (PH-LHD), which is the most common cause of PH. Paradoxically, lack of good preclinical model precludes extensive investigation of PH-LHD that remains dramatically understudied. Interestingly, recent epidemiologic studies showed a strong interconnection between metabolic syndrome (MetS) and heart failure with preserved ejection (HFpEF), which are two major public health issues in Western countries, and HP Group 2. First part of the project was to develop a new animal model that combines HFpEF (supracoronary aortic banding, SAB) and MetS (high-fat food, olanzapine), leading to PH development (right cardiac catheterization in closed chest) with increased pulmonary vascular remodelling. Then using our model, we display that PH is associated with ectopic fat (peripheral and mediastinal fat), increased plasmatic leptin-levels, and raised of pulmonary inflammation. Functionally we displayed that increased leptin-secretion from adipocytes is associated with STAT3 phosphorylation and pulmonary arterial smooth muscle cells (PASMC) proliferation of PH animals. Using in vitro experiments, we demonstrated that STAT3-activation induced by leptin is associated with PASMC proliferation. We also showed that leptin induces inflammatory cytokines secretion from activated macrophages. Finally, we displayed that leptin/STAT3 pathway contributes to the development of PH associated with HFpEF and MetS. To conclude, these works aimed to move away from the classical etiology of PH and introduce systemic impairments as important actors of the development and progression of PH, that should be considered as a potential target in the upcoming therapeutics

Right Ventricle and Epigenetics: A Systematic Review

There is an increasing recognition of the crucial role of the right ventricle (RV) in determining the functional status and prognosis in multiple conditions. In the past decade, the epigenetic regulation (DNA methylation, histone modification, and non-coding RNAs) of gene expression has been raised as a critical determinant of RV development, RV physiological function, and RV pathological dysfunction. We thus aimed to perform an up-to-date review of the literature, gathering knowledge on the epigenetic modifications associated with RV function/dysfunction. Therefore, we conducted a systematic review of studies assessing the contribution of epigenetic modifications to RV development and/or the progression of RV dysfunction regardless of the causal pathology. English literature published on PubMed, between the inception of the study and 1 January 2023, was evaluated. Two authors independently evaluated whether studies met eligibility criteria before study results were extracted. Amongst the 817 studies screened, 109 studies were included in this review, including 69 that used human samples (e.g., RV myocardium, blood). While 37 proposed an epigenetic-based therapeutic intervention to improve RV function, none involved a clinical trial and 70 are descriptive. Surprisingly, we observed a substantial discrepancy between studies investigating the expression (up or down) and/or the contribution of the same epigenetic modifications on RV function or development. This exhaustive review of the literature summarizes the relevant epigenetic studies focusing on RV in human or preclinical setting

Competition for oxygen among oxidative systems during bread dough mixing: consequences of addition of glucose oxidase and lipoxygenase on yeasted dough rheology

International audienceThe effect on O(2) uptake during the mixing of yeasted dough, either unsupplemented or supplemented with glucose oxidase (GOX), horsebean flour (HB), soybean flour (SB), or combinations thereof, was studied using an airtight mixer. Two wheat flours with a low (flour A) and a high (flour B) content of free polyunsaturated fatty acids were used. Addition of HB or SB provokes a similar increase of O(2) uptake for both wheat flours, whereas addition of GOX causes a larger increase for flour A than for flour B. When the wheat flours were supplemented with HB or SB, addition of GOX caused a small but significant increase of O(2) uptake for flour A. This increase was not observed for flour B. The mixing tolerance of dough A, determined with the Chopin Consistograph, is increased by GOX addition. However, this effect is less pronounced when flour A is supplemented with HB or SB. Similarly, the relaxation index of dough B is decreased by GOX addition, but the decrease is less distinct in the presence of HB or SB. These results can be explained by a competition among yeast, GOX, and lipoxygenases (present in wheat, HB, and SB flours) for the O(2) uptake by dough, which likely decreases the amount of hydrogen peroxide produced by GOX during dough mixing. This competition for O(2) consequently also modifies the rheological properties of dough

Supra-coronary aortic banding improves right ventricular function in experimental pulmonary arterial hypertension in rats by increasing systolic right coronary artery perfusion

Aim -- Pulmonary arterial hypertension (PAH) results in right ventricular (RV) dysfunction owing, in part, to RV ischemia. The relative contribution of RV microvascular rarefaction vs reduced right coronary artery perfusion pressure (RCA-PP) to RV ischemia remains unknown. We hypothesize that increasing RCA-PP improves RV function in PAH by increasing RV systolic perfusion. Methods -- Supra-coronary aortic banding (SAB) or sham surgery was performed on male Sprague-Dawley rats. Seven to ten days later, rats received either monocrotaline (MCT; 60 mg/kg) or saline. After 1 month, echocardiography, cardiac catheterization, 99mTc-sestamibi single-photon emission computed tomography (SPECT) and microsphere infusion studies were performed. The RV was harvested for measurement of hypertrophy (RVH), fibrosis and immunoblotting, and the lung was harvested for pulmonary artery (PA) histology. Results -- Supra-coronary aortic banding increased systolic pressures in proximal aorta and systolic RCA-PP in SAB + MCT vs MCT rats (114 ± 12 vs 5 ± 9 mm Hg), without altering diastolic RCA-PP. SAB + MCT rats had improved RV function vs MCT rats, evident from their significantly increased cardiac output (CO), RV free wall (RVFW) thickening, tricuspid annular plane systolic excursion (TAPSE) and RV-PA coupling indices. RV-PA coupling indices and CO correlated directly with systolic RCA-PP. RV perfusion was increased in SAB + MCT vs MCT rats and correlated well with CO; whereas microvascular rarefaction was unaltered. SAB + MCT rats had less RVH and fibrosis and lower PA pressures vs MCT rats. SAB + MCT rats had significantly lower RV pyruvate kinase muscle isoform 2/1 ratios than MCT rats, consistent with restoration of oxidative metabolism. Conclusion -- A SAB-induced increase in systolic RCA-PP improves RV perfusion and function in MCT rats. Maintaining systolic RCA perfusion can preserve RV function in PAH

Inhibiting pyruvate kinase muscle isoform 2 regresses group 2 pulmonary hypertension induced by supra-coronary aortic banding

Introduction: Group 2 pulmonary hypertension (PH) has no approved PH-targeted therapy. Metabolic remodelling, specifically a biventricular increase in pyruvate kinase muscle (PKM) isozyme 2 to 1 ratio, occurs in rats with group 2 PH induced by supra-coronary aortic banding (SAB). We hypothesize that increased PKM2/PKM1 is maladaptive and inhibiting PKM2 would improve right ventricular (RV) function. Methods: Male, Sprague-Dawley SAB rats were confirmed to have PH by echocardiography and then randomized to treatment with a PKM2 inhibitor (intraperitoneal shikonin, 2 mg/kg/day) versus 5% DMSO (n = 5/group) or small interfering RNA-targeting PKM2 (siPKM2) versus siRNA controls (n = 7/group) by airway nebulization. Results: Shikonin-treated SAB rats had milder PH (PAAT 32.1 ± 1.3 vs 22.1 ± 1.2 ms, P =.0009) and lower RV systolic pressure (RVSP) (31.5 ± 0.9 vs 55.7 ± 1.9 mm Hg, P <.0001) versus DMSO-SAB rats. siPKM2 nebulization reduced PKM2 expression in the RV, increased PAAT (31.7 ± 0.7 vs 28.0 ± 1.3 ms, P =.025), lowered RVSP (30.6 ± 2.6 vs 42.0 ± 4.0 mm Hg, P =.032) and reduced diastolic RVFW thickness (0.69 ± 0.04 vs 0.85 ± 0.06 mm, P =.046). Both shikonin and siPKM2 regressed PH-induced medial hypertrophy of small pulmonary arteries. Conclusion: Increases in PKM2/PKM1 in the RV contribute to RV dysfunction in group 2 PH. Chemical or molecular inhibition of PKM2 restores the normal PKM2/PKM1 ratio, reduces PH, RVSP and RVH and regresses adverse PA remodelling. PKM2 merits consideration as a therapeutic cardiac target for group 2 PH

Mitochondria in the pulmonary vasculature in health and disease : oxygen-sensing, metabolism, and dynamics

In lung vascular cells, mitochondria serve a canonical metabolic role, governing energy homeostasis. In addition, mitochondria exist in dynamic networks, which serve noncanonical functions, including regulation of redox signaling, cell cycle, apoptosis, and mitochondrial quality control. Mitochondria in pulmonary artery smooth muscle cells (PASMC) are oxygen sensors and initiate hypoxic pulmonary vasoconstriction. Acquired dysfunction of mitochondrial metabolism and dynamics contribute to a cancer‐like phenotype in pulmonary arterial hypertension (PAH). Acquired mitochondrial abnormalities, such as increased pyruvate dehydrogenase kinase (PDK) and pyruvate kinase muscle isoform 2 (PKM2) expression, which increase uncoupled glycolysis (the Warburg phenomenon), are implicated in PAH. Warburg metabolism sustains energy homeostasis by the inhibition of oxidative metabolism that reduces mitochondrial apoptosis, allowing unchecked cell accumulation. Warburg metabolism is initiated by the induction of a pseudohypoxic state, in which DNA methyltransferase (DNMT)‐mediated changes in redox signaling cause normoxic activation of HIF‐1α and increase PDK expression. Furthermore, mitochondrial division is coordinated with nuclear division through a process called mitotic fission. Increased mitotic fission in PAH, driven by increased fission and reduced fusion favors rapid cell cycle progression and apoptosis resistance. Downregulation of the mitochondrial calcium uniporter complex (MCUC) occurs in PAH and is one potential unifying mechanism linking Warburg metabolism and mitochondrial fission. Mitochondrial metabolic and dynamic disorders combine to promote the hyperproliferative, apoptosis‐resistant, phenotype in PAH PASMC, endothelial cells, and fibroblasts. Understanding the molecular mechanism regulating mitochondrial metabolism and dynamics has permitted identification of new biomarkers, nuclear and CT imaging modalities, and new therapeutic targets for PAH

HDAC6: A Novel Histone Deacetylase Implicated in Pulmonary Arterial Hypertension

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 "cancer-like" pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase regulating 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. We found that HDAC6 is significantly up-regulated in lungs, distal PAs, and isolated PASMCs from PAH patients and animal models. Inhibition of HDAC6 reduced PAH-PASMC proliferation and resistance to apoptosis in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 maintains Ku70 in a hypoacetylated state, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo, pharmacological inhibition of HDAC6 improved established PAH in two experimental models and can be safely given in combination with currently approved PAH therapies. Moreover, Hdac6 deficient mice were partially protected against chronic hypoxia-induced pulmonary hypertension. Our study shows for the first time that HDAC6 is implicated in PAH development and represents a new promising target to improve PAH

Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension

Background—Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. Methods and Results—We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription–polymerase chain reaction; P<0.01) and capillary density (CD31+ immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. Conclusions—RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH

Downregulation of microRNA-126 contributes to the failing right ventricle in pulmonary arterial hypertension

Background—Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. Methods and Results—We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription–polymerase chain reaction; P<0.01) and capillary density (CD31+ immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. Conclusions—RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH