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BACKGROUND: Pulmonary arterial hypertension (PAH) ultimately leads to right ventricular failure and premature death. The identification of circulating biomarkers with prognostic utility is considered a priority. As chronic inflammation is recognized as key pathogenic driver, we sought to identify inflammation-related circulating proteins that add incremental value to current risk stratification models for long-term survival in patients with PAH. METHODS AND RESULTS: Plasma levels of 384 inflammatory proteins were measured with the proximity extension assay technology in patients with PAH (n=60) and controls with normal hemodynamics (n=28). Among these, 51 analytes were significantly overexpressed in the plasma of patients with PAH compared with controls. Cox proportional hazard analyses and C-statistics were performed to assess the prognostic value and the incremental prognostic value of differentially expressed proteins. A panel of 6 proteins (CRIM1 [cysteine rich transmembrane bone morphogenetic protein regulator 1], HGF [hepatocyte growth factor], FSTL3 [follistatin-like 3], PLAUR [plasminogen activator, urokinase receptor], CLSTN2 [calsyntenin 2], SPON1 [spondin 1]) were independently associated with death/lung transplantation at the time of PAH diagnosis after adjustment for the 2015 European Society of Cardiology/European Respiratory Society guidelines, the REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) 2.0 risk scores, and the refined 4-strata risk assessment. CRIM1, PLAUR, FSTL3, and SPON1 showed incremental prognostic value on top of the predictive models. As determined by Western blot, FSTL3 and SPON1 were significantly upregulated in the right ventricle of patients with PAH and animal models (monocrotaline-injected and pulmonary artery banding-subjected rats). CONCLUSIONS: In addition to revealing new actors likely involved in cardiopulmonary remodeling in PAH, our screening identified promising circulating biomarkers to improve risk prediction in PAH, which should be externally confirmed.
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Biomarcadores , Proteómica , Hipertensión Arterial Pulmonar , Humanos , Masculino , Femenino , Biomarcadores/sangre , Proteómica/métodos , Persona de Mediana Edad , Pronóstico , Hipertensión Arterial Pulmonar/sangre , Hipertensión Arterial Pulmonar/mortalidad , Hipertensión Arterial Pulmonar/diagnóstico , Hipertensión Arterial Pulmonar/fisiopatología , Adulto , Animales , Medición de Riesgo , Estudios de Casos y Controles , Receptores del Activador de Plasminógeno Tipo Uroquinasa/sangre , Proteínas Relacionadas con la Folistatina/sangre , Modelos Animales de Enfermedad , Valor Predictivo de las Pruebas , Inflamación/sangre , Mediadores de Inflamación/sangre , Factores de Riesgo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/sangre , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Arteria Pulmonar/fisiopatologíaRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
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BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.
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Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt , Ubiquitina Tiolesterasa , Animales , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/deficiencia , Ubiquitina Tiolesterasa/metabolismo , Humanos , Ratones , Ratas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Masculino , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/genética , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/enzimología , Ratas Sprague-Dawley , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/etiología , Remodelación Vascular , Células Cultivadas , Proliferación Celular , Ratones Endogámicos C57BL , Indoles , OximasRESUMEN
Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.
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Hipertensión Arterial Pulmonar , Transducción de Señal , Remodelación Vascular , Animales , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/fisiología , Ratas , Humanos , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Hipertensión Arterial Pulmonar/metabolismo , Masculino , Modelos Animales de Enfermedad , Arteria Pulmonar/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Ratones , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción , Proliferación Celular/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/metabolismo , Lisina/análogos & derivadosRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.
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Hipertensión Arterial Pulmonar , Ratas , Ratones , Animales , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Remodelación Vascular , Proliferación Celular , Hipertensión Pulmonar Primaria Familiar , Modelos Animales de Enfermedad , Miocitos del Músculo Liso , Arteria PulmonarRESUMEN
Right ventricular (RV) function is critical to prognosis in all forms of pulmonary hypertension. Here we perform molecular phenotyping of RV remodeling by transcriptome analysis of RV tissue obtained from 40 individuals, and two animal models of RV dysfunction of both sexes. Our unsupervised clustering analysis identified 'early' and 'late' subgroups within compensated and decompensated states, characterized by the expression of distinct signaling pathways, while fatty acid metabolism and estrogen response appeared to underlie sex-specific differences in RV adaptation. The circulating levels of several extracellular matrix proteins deregulated in decompensated RV subgroups were assessed in two independent cohorts of individuals with pulmonary arterial hypertension, revealing that NID1, C1QTNF1 and CRTAC1 predicted the development of a maladaptive RV state, as defined by magnetic resonance imaging parameters, and were associated with worse clinical outcomes. Our study provides a resource for subphenotyping RV states, identifying state-specific biomarkers, and potential therapeutic targets for RV dysfunction.
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Perfilación de la Expresión Génica , Disfunción Ventricular Derecha , Función Ventricular Derecha , Remodelación Ventricular , Masculino , Humanos , Femenino , Remodelación Ventricular/genética , Remodelación Ventricular/fisiología , Animales , Función Ventricular Derecha/fisiología , Disfunción Ventricular Derecha/genética , Disfunción Ventricular Derecha/fisiopatología , Persona de Mediana Edad , Modelos Animales de Enfermedad , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/metabolismo , Transcriptoma , Adaptación Fisiológica , Factores Sexuales , Adulto , FenotipoRESUMEN
BACKGROUND: We explored the mechanism of maladaptive right ventricular (RV) remodeling in Fischer compared with Sprague-Dawley (SD) rats exposed to pressure overload. METHODS: Pulmonary hypertension was induced by injection of the VEGFR antagonist, SU5416, followed by a 3-week exposure to hypoxia (Sugen chronic hypoxia). In vivo oxidative metabolism was assessed by RV/left ventricle ratio of [11C]acetate positron emission tomography clearance (kmono). Unbiased, global transcriptional and proteomic profiling was performed in Fischer and SD rats at baseline and after Sugen chronic hypoxia. RESULTS: All Fischer rats succumbed to RV failure by 5 weeks, whereas SD rats showed preserved RV function and 88% survival beyond 9 weeks (P<0.0001). Fischer rats exhibited increased oxidative metabolism at 4 weeks (P<0.05) and impaired RV efficiency compared with SD (work metabolic index: 52±10 versus 91±27 mmHg·mL/cm2, respectively; P<0.05), but no differences in mitochondrial complex activity. AK1 (adenylate kinase 1) was among the top 10 differentially expressed genes between Fischer and SD rats, with markedly lower RV expression in Fischer rats (FC: 3.36, P<0.05), confirmed by proteomic analysis and validated by Western blotting (>10-fold reduction, P<0.001). While whole-genome sequencing failed to reveal any coding region mutations in Fischer rats, there was a unique variant in a highly conserved upstream flanking region likely involved in the regulation of AK1 expression. CONCLUSIONS: Therefore, Fischer rats exhibit profound AK1 deficiency and inefficient cardiac energetics likely related to reduced adenosine triphosphate shuttling from the mitochondria to the contractile fibers. This represents a novel mechanism for RV failure in response to chronic increases in afterload.
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Insuficiencia Cardíaca , Ventrículos Cardíacos , Ratas , Animales , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Proteómica , Función Ventricular Derecha , Remodelación Ventricular , Hipoxia/metabolismo , Modelos Animales de EnfermedadRESUMEN
Background: The blood neutrophil-to-lymphocyte ratio (NLR) has recently emerged as a powerful predictor of adverse outcomes in some cardiovascular and lung diseases. Pulmonary arterial hypertension (PAH) is a lethal vasculopathy associated with increased inflammation. Although PAH exhibits a higher prevalence among women, men have a poorer prognosis. We investigated the NLR as an independent predictor of transplant-free survival in PAH. Methods: We performed a retrospective analysis of 78 PAH patients from the Quebec PAHBiobank (71% female). We used univariate and multivariate (adjusted for age, sex, renal function, and disease severity) Cox regression analyses to assess the relationship between the NLR and transplant-free survival, in the whole sample, and according to sex. The NLR was categorized as high (≥ 4.8) or low (< 4.8) using receiver operating characteristic analysis. Unadjusted Kaplan-Meier analysis estimated survival per NLR category. Results: The NLR was higher in patients who died, compared to that in patients who had transplant-free survival (P < 0.05). The NLR was an independent predictor of event-free survival in PAH (unadjusted hazard ratio: 1.11, 95% confidence interval: 1.04-1.18, which remained significant after adjustment for covariates). The high-NLR group had lower 1-, 3-, and 5-year survival compared to those with a low NLR (P < 0.001). The NLR remains a predictor of survival in women. Conclusions: The NLR is an independent predictor of transplant-free survival in PAH. We report a potential sexual dimorphism in the ability of the NLR to predict mortality in PAH, emphasizing the importance of considering sex-related differences in the development of biomarkers in PAH.
Contexte: Le rapport neutrophiles/lymphocytes (RNL) s'est récemment imposé comme un puissant facteur prédictif de l'issue défavorable de certaines maladies cardiovasculaires et pulmonaires. L'hypertension artérielle pulmonaire (HTAP) est une vasculopathie mortelle associée à une inflammation accrue. Bien que sa prévalence soit plus élevée chez les femmes, son pronostic est plus défavorable chez les hommes. Nous avons étudié le RNL en tant que prédicteur indépendant de la survie sans greffe chez des sujets atteints d'HTAP. Méthodologie: Nous avons effectué une analyse rétrospective du matériel sur l'HTAP de la Biobanque du Québec se rapportant à 78 patients (71 % de femmes). Des analyses de régression de Cox univariées et multivariées (ajustées en fonction de l'âge, du sexe, de la fonction rénale et de la gravité de la maladie) nous ont permis d'évaluer la relation entre le RNL et la survie sans greffe dans l'ensemble de l'échantillon et selon le sexe. Le RNL a été classé comme élevé (≥ 4,8) ou faible (< 4,8) au terme d'une analyse des caractéristiques de fonctionnement du récepteur. Une analyse non ajustée effectuée selon la méthode de Kaplan-Meier a servi à estimer la survie par catégorie de RNL. Résultats: Le RNL était plus élevé chez les patients décédés que chez les patients ayant survécu sans greffe (p < 0,05). Le RNL était un prédicteur indépendant de la survie sans événement chez les patients atteints d'HTAP (rapport des risques instantanés non ajusté : 1,11, intervalle de confiance à 95 % : 1,04-1,18, demeuré significatif après ajustement en fonction des covariables). La survie à 1, 3 et 5 ans a été inférieure au sein du groupe présentant un RNL élevé comparativement au groupe présentant un RNL faible (p < 0,001). Le RNL demeure un prédicteur de la survie chez les femmes. Conclusions: Le RNL est un facteur prédictif indépendant de la survie sans greffe chez les sujets atteints d'HTAP. Selon nos observations, un dimorphisme sexuel potentiel le caractérise en tant que prédicteur de la mortalité chez les sujets atteints d'HTAP. Il importe donc de tenir compte des différences liées au sexe dans le développement des biomarqueurs de l'HTAP.
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The authors show that increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) and pyruvate kinase muscle isozyme 2 (PKM2) expression is a common feature of a decompensated right ventricle in patients with pulmonary arterial hypertension and animal models. The authors find in vitro that overactivated PARP1 promotes cardiomyocyte dysfunction by favoring PKM2 expression and nuclear function, glycolytic gene expression, activation of nuclear factor κB-dependent proinflammatory factors. Pharmacologic and genetic inhibition of PARP1 or enforced tetramerization of PKM2 attenuates maladaptive remodeling improving right ventricular (RV) function in multiple rodent models. Taken together, these data implicate the PARP1/PKM2 axis as a critical driver of maladaptive RV remodeling and a new promising target to directly sustain RV function in patients with pulmonary arterial hypertension.
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RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hipertensión Pulmonar , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Hipertensión Arterial Pulmonar , Animales , Proliferación Celular , Células Cultivadas , Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , Mamíferos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Hipertensión Arterial Pulmonar/genética , Arteria Pulmonar/metabolismo , Remodelación Vascular/fisiologíaRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterised by exuberant tissue remodelling and associated with high unmet medical needs. Outcomes are even worse when IPF results in secondary pulmonary hypertension (PH). Importantly, exaggerated resistance to cell death, excessive proliferation and enhanced synthetic capacity are key endophenotypes of both fibroblasts and pulmonary artery smooth muscle cells, suggesting shared molecular pathways. Under persistent injury, sustained activation of the DNA damage response (DDR) is integral to the preservation of cells survival and their capacity to proliferate. Checkpoint kinases 1 and 2 (CHK1/2) are key components of the DDR. The objective of this study was to assess the role of CHK1/2 in the development and progression of IPF and IPF+PH. METHODS AND RESULTS: Increased expression of DNA damage markers and CHK1/2 were observed in lungs, remodelled pulmonary arteries and isolated fibroblasts from IPF patients and animal models. Blockade of CHK1/2 expression or activity-induced DNA damage overload and reverted the apoptosis-resistant and fibroproliferative phenotype of disease cells. Moreover, inhibition of CHK1/2 was sufficient to interfere with transforming growth factor beta 1-mediated fibroblast activation. Importantly, pharmacological inhibition of CHK1/2 using LY2606368 attenuated fibrosis and pulmonary vascular remodelling leading to improvement in respiratory mechanics and haemodynamic parameters in two animal models mimicking IPF and IPF+PH. CONCLUSION: This study identifies CHK1/2 as key regulators of lung fibrosis and provides a proof of principle for CHK1/2 inhibition as a potential novel therapeutic option for IPF and IPF+PH.
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Hipertensión Pulmonar , Fibrosis Pulmonar Idiopática , Animales , Fibroblastos/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Although right ventricular (RV) function is the primary determinant of morbidity and mortality in pulmonary arterial hypertension (PAH), the molecular mechanisms of RV remodeling and the circulating factors reflecting its function remain largely elusive. In this context, the identification of new molecular players implicated in maladaptive RV remodeling along with the optimization of risk stratification approaches in PAH are key priorities. Through combination of transcriptomic and proteomic profiling of RV tissues with plasma proteome profiling, we identified a panel of proteins, mainly related to cardiac fibrosis, similarly upregulated in the RV and plasma of patients with PAH with decompensated RV. Among these, we demonstrated that plasma latent transforming growth factor beta binding protein 2 (LTBP-2) level correlates with RV function in human PAH and adds incremental value to current risk stratification models to predict long-term survival in two independent PAH cohorts.
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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.
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Proteína Potenciadora del Homólogo Zeste 2/genética , Proteoma/genética , Hipertensión Arterial Pulmonar/genética , Transcriptoma/genética , Animales , Apoptosis/genética , Proliferación Celular/genética , Ciclo del Ácido Cítrico/genética , Epigénesis Genética/genética , Femenino , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/crecimiento & desarrollo , Arteria Pulmonar/patología , RatasRESUMEN
Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/prevención & control , Miocitos del Músculo Liso/efectos de los fármacos , Trifluoperazina/farmacología , Animales , Antipsicóticos/farmacología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Hemodinámica/efectos de los fármacos , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Hipoxia/inducido químicamente , Hipoxia/genética , Hipoxia/fisiopatología , Indoles/administración & dosificación , Monocrotalina/administración & dosificación , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Pirroles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Survivin/genética , Survivin/metabolismoRESUMEN
Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.
Asunto(s)
Apelina/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Hipertensión Pulmonar/fisiopatología , Función Ventricular Derecha/fisiología , Animales , Cardiotónicos/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Ratas , Ratas MutantesRESUMEN
Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.