RESUMEN
BACKGROUND: Despite recent advances the prognosis of pulmonary hypertension remains poor and warrants novel therapeutic options. Extensive studies, including ours, have revealed that hypoxia-induced pulmonary hypertension is associated with high oxidative stress. Cerium oxide nanozyme or nanoparticles (CeNPs) have displayed catalytic activity mimicking both catalase and superoxide dismutase functions and have been widely used as an anti-oxidative stress approach. However, whether CeNPs can attenuate hypoxia-induced pulmonary vascular oxidative stress and pulmonary hypertension is unknown. RESULTS: In this study, we designed a new ceria nanozyme or nanoparticle (AuCeNPs) exhibiting enhanced enzyme activity. The AuCeNPs significantly blunted the increase of reactive oxygen species and intracellular calcium concentration while limiting proliferation of pulmonary artery smooth muscle cells and pulmonary vasoconstriction in a model of hypoxia-induced pulmonary hypertension. In addition, the inhalation of nebulized AuCeNPs, but not CeNPs, not only prevented but also blunted hypoxia-induced pulmonary hypertension in rats. The benefits of AuCeNPs were associated with limited increase of intracellular calcium concentration as well as enhancement of extracellular calcium-sensing receptor (CaSR) activity and expression in rat pulmonary artery smooth muscle cells. Nebulised AuCeNPs showed a favorable safety profile, systemic arterial pressure, liver and kidney function, plasma Ca2+ level, and blood biochemical parameters were not affected. CONCLUSION: We conclude that AuCeNPs is an improved reactive oxygen species scavenger that effectively prevents and treats hypoxia-induced pulmonary hypertension.
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Cerio , Hipertensión Pulmonar , Hipoxia , Miocitos del Músculo Liso , Arteria Pulmonar , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Animales , Cerio/farmacología , Cerio/química , Cerio/uso terapéutico , Ratas , Hipertensión Pulmonar/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Masculino , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Nanopartículas/química , Calcio/metabolismoRESUMEN
INTRODUCTION: Aberrant gene expression is a key mechanism underlying pulmonary hypertension (PH) development. The alterations of genomic chromatin accessibility and their relationship with the aberrant gene expressions in PH are poorly understood. We used bulk Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) in pulmonary artery smooth muscle cells (PASMCs) of chronic hypoxia-exposed rats mimicking group 3 human PH. METHODS: Adult Sprague Dawley rats were commercially obtained from Hunan SJA (Hunan SJA Laboratory Animal Co., Changsha, China) and randomizedly allocated into four groups exposing to nomobaric hypoxia or normoxia for 1 or 28 days respectively. After the assessment of pulmonary hemodynamics, smooth muscle cells were isolated from intralobular arteries and simultaneously subjected to bulk Assay of ATAC-seq and RNA-seq. RESULTS: Hypoxic exposure for continuous 28-days, but not for 1-day, induced established PH phenotypes in rats. ATAC-seq revealed a major distribution of differential accessibility regions (DARs) annotated to the genome in out-of-promoter regions, following 1-day or 28-days hypoxia. 1188 DAR-associated genes and 378 differentially expressed genes (DEGs) were identified in rats after exposure to 1-day hypoxia, while 238 DAR-associated genes and 452 DEGs for 28-days hypoxia. Most of the DAR-associated genes or DEGs in 1-day did not overlap with that of 28-days hypoxia. A Pearson correlation analysis indicated no significant correlation between ATAC-seq and RNA-seq. CONCLUSIONS: The alterations in genomic chromatin accessibility and genes expression of PASMCs in the initial stage of hypoxia are distinct from the established stage of hypoxia-induced PH. The genomic differential accessibility regions may not be the main mechanisms directly underlying the differentially expressed genes observed either in the initial or established stages of PH. Thus the time-course alterations of gene expression and their possible indirect link with genomic chromatin accessibility warrant more attention in mechanistic study of pulmonary hypertension.
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Cromatina , Hipertensión Pulmonar , Adulto , Animales , Humanos , Ratas , Cromatina/genética , Hipertensión Pulmonar/genética , Ratas Sprague-Dawley , Hipoxia/genética , Hipoxia/complicaciones , Genómica , Expresión GénicaRESUMEN
Venous thromboembolism (VTE) is a multifactorial disease, and pulmonary hypertension (PH) is a serious condition characterized by pulmonary vascular remodeling leading with increased pulmonary vascular resistance, ultimately leading to right heart failure and death. Although VTE and PH have distinct primary etiologies, they share some pathophysiologic similarities such as dysfunctional vasculature and thrombosis. In both conditions there is solid evidence that EVs derived from a variety of cell types including platelets, monocytes, endothelial cells and smooth muscle cells contribute to vascular endothelial dysfunction, inflammation, thrombosis, cellular activation and communications. However, the roles and importance of EVs substantially differ between studies depending on experimental conditions and parent cell origins of EVs that modify the nature of their cargo. Numerous studies have confirmed that EVs contribute to the pathophysiology of VTE and PH and increased levels of various EVs in relation with the severity of VTE and PH, confirming its potential pathophysiological role and its utility as a biomarker of disease severity and as potential therapeutic targets.
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Vesículas Extracelulares , Hipertensión Pulmonar , Trombosis , Tromboembolia Venosa , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/terapia , Tromboembolia Venosa/metabolismo , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
BACKGROUND: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. METHODS: We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. RESULTS: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. CONCLUSIONS: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Fosfatos de Poliisoprenilo/efectos adversos , Animales , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Masculino , RatasRESUMEN
Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.
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Aterosclerosis , Diabetes Mellitus Tipo 2 , Cardiomiopatías Diabéticas , Insuficiencia Cardíaca , Infarto del Miocardio , Animales , Diabetes Mellitus Tipo 2/complicaciones , Cardiomiopatías Diabéticas/complicaciones , Insuficiencia Cardíaca/etiología , Humanos , Hipoglucemiantes , Infarto del Miocardio/complicacionesRESUMEN
Phenylalanine levels are associated with pulmonary hypertension in metabolic profiling clinical studies. However, the pathophysiological role of phenylalanine on pulmonary circulation is still unclear. We experimentally addressed the direct impact of phenylalanine on pulmonary circulation in rats and explored the underlying molecular pathway. Phenylalanine was injected intraperitoneally into Sprague-Dawley rats (400 mg/100 g body wt) as a single dose or daily in a chronic manner for 2, 3, and 4 wk. Chronic injection of phenylalanine induced pulmonary hypertension with time-dependent severity, evidenced by elevated pulmonary artery pressure and pulmonary vascular resistance as well as pulmonary artery and right ventricular hypertrophy. Using tandem mass spectrometry analysis, we found a quick twofold increase in blood level of phenylalanine 2 h following injection. This increase led to a significant accumulation of phenylalanine in lung after 4 h, which remained sustained at up to a threefold increase after 4 wk. In addition, a cellular thermal shift assay with lung tissues from phenylalanine-injected rats revealed the binding of phenylalanine to the calcium-sensing receptor (CaSR). In vitro experiments with cultured pulmonary arterial smooth muscle cells showed that phenylalanine activated CaSR, as indicated by an increase in intracellular calcium content, which was attenuated or diminished by the inhibition or knockdown of CaSR. Finally, the global knockout or lung-specific knockdown of CaSR significantly attenuated phenylalanine-induced pulmonary hypertension. Chronic phenylalanine injection induces pulmonary hypertension through binding to CaSR and its subsequent activation. Here, we demonstrate a pathophysiological role of phenylalanine in pulmonary hypertension through the CaSR. This study provides a novel animal model for pulmonary hypertension and reveals a potentially clinically significant role for this metabolite in human pulmonary hypertension as a marker, a mediator of disease, and a possible therapeutic target.
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Señalización del Calcio/efectos de los fármacos , Hipertensión Pulmonar/metabolismo , Fenilalanina/farmacología , Receptores Sensibles al Calcio/efectos de los fármacos , Animales , Señalización del Calcio/fisiología , Hipertensión Pulmonar/inducido químicamente , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Ratas Sprague-Dawley , Receptores Sensibles al Calcio/metabolismoRESUMEN
Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.
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Deficiencia de Citocromo-c Oxidasa/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Fibroblastos/enzimología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedad de Leigh/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Neoplasias/metabolismo , Piel/enzimología , Adenosina Trifosfato/metabolismo , Células Cultivadas , Niño , Deficiencia de Citocromo-c Oxidasa/genética , Deficiencia de Citocromo-c Oxidasa/patología , Complejo IV de Transporte de Electrones/genética , Metabolismo Energético , Femenino , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mitocondrias/enzimología , Mitocondrias/patología , Proteínas de Neoplasias/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Quebec , Transducción de Señal , Piel/patologíaRESUMEN
The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.
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Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Permeabilidad de la Membrana Celular/genética , Deficiencia de Citocromo-c Oxidasa/genética , Deficiencia de Citocromo-c Oxidasa/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético , Femenino , Hepatocitos/metabolismo , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Hígado/metabolismo , Masculino , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Fosforilación Oxidativa , Poliadenilación , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN MitocondrialRESUMEN
The goal of this work was to develop a label-free, comprehensive, and reproducible high-resolution liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomic workflow using a single instrument, which could be applied to biomarker discovery in both basic and clinical studies. For this, we have (i) optimized lipid extraction and elution to enhance coverage of polar and nonpolar lipids as well as resolution of their isomers, (ii) ensured MS signal reproducibility and linearity, and (iii) developed a bioinformatic pipeline to correct remaining biases. Workflow validation is reported for 48 replicates of a single human plasma sample: 1124 reproducible LC-MS signals were extracted (median signal intensity RSD = 10%), 50% of which are redundant due to adducts, dimers, in-source fragmentation, contaminations, or positive and negative ion duplicates. From the resulting 578 unique compounds, 428 lipids were identified by MS/MS, including acyl chain composition, of which 394 had RSD < 30% inside their linear intensity range, thereby enabling robust semiquantitation. MS signal intensity spanned 4 orders of magnitude, covering 16 lipid subclasses. Finally, the power of our workflow is illustrated by a proof-of-concept study in which 100 samples from healthy human subjects were analyzed and the data set was investigated using three different statistical testing strategies in order to compare their capacity in identifying the impact of sex and age on circulating lipids.
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Cromatografía Liquida/métodos , Lípidos/aislamiento & purificación , Metaboloma/fisiología , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/instrumentación , Biología Computacional/métodos , Voluntarios Sanos , Humanos , Lípidos/sangre , Lípidos/química , Lípidos/clasificación , Anotación de Secuencia Molecular , Análisis de Componente Principal , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Various signaling pathways have been identified in the heart as important players during development, physiological adaptation or pathological processes. This includes the MAPK families, particularly p38MAPK, which is involved in several key cellular processes, including differentiation, proliferation, apoptosis, inflammation, metabolism and survival. Disrupted p38MAPK signaling has been associated with several diseases, including cardiovascular diseases (CVD) as well as diabetes and its related complications. Despite efforts to translate this knowledge into therapeutic avenues, p38 inhibitors have failed in clinical trials due to adverse effects. Inhibition of MK2, a downstream target of p38, appears to be a promising alternative strategy. Targeting MK2 activity may avoid the adverse effects linked to p38 inhibition, while maintaining its beneficial effects. MK2 was first considered as a therapeutic target in inflammatory diseases such as rheumatoid polyarthritis. A growing body of evidence now supports a key role of MK2 signaling in the pathogenesis of CVD, particularly ischemia/reperfusion injury, hypertrophy, and hypertension and that its inhibition or inactivation is associated with improved heart and vascular functions. More recently, MK2 was shown to be a potential player in diabetes and related complications, particularly in liver and heart, and perturbations in calcium handling and lipid metabolism. In this review, we will discuss recent advances in our knowledge of the role of MK2 in p38MAPK-mediated signaling and the benefits of its loss of function in CVD and diabetes, with an emphasis on the roles of MK2 in calcium handling and lipid metabolism. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.
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Cardiomiopatías Diabéticas/enzimología , Metabolismo Energético , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos , Miocitos Cardíacos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Fármacos Cardiovasculares/uso terapéutico , Cardiomiopatías Diabéticas/tratamiento farmacológico , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/fisiopatología , Metabolismo Energético/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Metabolismo de los Lípidos/efectos de los fármacos , Terapia Molecular Dirigida , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Heart failure (HF) is associated with metabolic perturbations, particularly of fatty acids (FAs), which remain to be better understood in humans. This study aimed at testing the hypothesis that HF patients with reduced ejection fraction display systemic perturbations in levels of energy-related metabolites, especially those reflecting dysregulation of FA metabolism, namely, acylcarnitines (ACs). Circulating metabolites were assessed using mass spectrometry (MS)-based methods in two cohorts. The main cohort consisted of 72 control subjects and 68 HF patients exhibiting depressed left ventricular ejection fraction (25.9 ± 6.9%) and mostly of ischemic etiology with ≥2 comorbidities. HF patients displayed marginal changes in plasma levels of tricarboxylic acid cycle-related metabolites or indexes of mitochondrial or cytosolic redox status. They had, however, 22-79% higher circulating ACs, irrespective of chain length (P < 0.0001, adjusted for sex, age, renal function, and insulin resistance, determined by shotgun MS/MS), which reflects defective mitochondrial ß-oxidation, and were significantly associated with levels of NH2-terminal pro-B-type natriuretic peptide levels, a disease severity marker. Subsequent extended liquid chromatography-tandem MS analysis of 53 plasma ACs in a subset group from the primary cohort confirmed and further substantiated with a comprehensive lipidomic analysis in a validation cohort revealed in HF patients a more complex circulating AC profile. The latter included dicarboxylic-ACs and dihydroxy-ACs as well as very long chain (VLC) ACs or sphingolipids with VLCFAs (>20 carbons), which are proxies of dysregulated FA metabolism in peroxisomes. Our study identified alterations in circulating ACs in HF patients that are independent of biological traits and associated with disease severity markers. These alterations reflect dysfunctional FA metabolism in mitochondria but also beyond, namely, in peroxisomes, suggesting a novel mechanism contributing to global lipid perturbations in human HF.NEW & NOTEWORTHY Mass spectrometry-based profiling of circulating energy metabolites, including acylcarnitines, in two cohorts of heart failure versus control subjects revealed multiple alterations in fatty acid metabolism in peroxisomes in addition to mitochondria, thereby highlighting a novel mechanism contributing to global lipid perturbations in heart failure.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/acylcarnitines-in-human-heart-failure/.
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Carnitina/análogos & derivados , Ácidos Grasos/metabolismo , Insuficiencia Cardíaca/sangre , Trastornos del Metabolismo de los Lípidos/sangre , Mitocondrias Cardíacas/metabolismo , Anciano , Carnitina/sangre , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peroxisomas/metabolismo , Esfingolípidos/metabolismo , Volumen Sistólico , Disfunción Ventricular Izquierda/sangre , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
While heart rate reduction (HRR) is a target for the management of patients with heart disease, contradictory results were reported using ivabradine, which selectively inhibits the pacemaker If current, vs. ß-blockers like metoprolol. This study aimed at testing whether similar HRR with ivabradine vs. metoprolol differentially modulates cardiac energy substrate metabolism, a factor determinant for cardiac function, in a mouse model of dyslipidemia (hApoB+/+;LDLR-/-). Following a longitudinal study design, we used 3- and 6-mo-old mice, untreated or treated for 3 mo with ivabradine or metoprolol. Cardiac function was evaluated in vivo and ex vivo in working hearts perfused with 13C-labeled substrates to assess substrate fluxes through energy metabolic pathways. Compared with 3-mo-old, 6-mo-old dyslipidemic mice had similar cardiac hemodynamics in vivo but impaired (P < 0.001) contractile function (aortic flow: -45%; cardiac output: -34%; stroke volume: -35%) and glycolysis (-24%) ex vivo. Despite inducing a similar 10% HRR, ivabradine-treated hearts displayed significantly higher stroke volume values and glycolysis vs. their metoprolol-treated counterparts ex vivo, values for the ivabradine group being often not significantly different from 3-mo-old mice. Further analyses highlighted additional significant cardiac alterations with disease progression, namely in the total tissue level of proteins modified by O-linked N-acetylglucosamine (O-GlcNAc), whose formation is governed by glucose metabolism via the hexosamine biosynthetic pathway, which showed a similar pattern with ivabradine vs. metoprolol treatment. Collectively, our results emphasize the implication of alterations in cardiac glucose metabolism and signaling linked to disease progression in our mouse model. Despite similar HRR, ivabradine, but not metoprolol, preserved cardiac function and glucose metabolism during disease progression.
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Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Benzazepinas/farmacología , Fármacos Cardiovasculares/farmacología , Dislipidemias/metabolismo , Glucólisis/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Corazón/efectos de los fármacos , Metoprolol/farmacología , Animales , Bradicardia , Modelos Animales de Enfermedad , Ecocardiografía , Metabolismo Energético/efectos de los fármacos , Femenino , Glucosa/metabolismo , Hemodinámica/efectos de los fármacos , Ivabradina , Estudios Longitudinales , Masculino , Ratones , Miocardio/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Volumen Sistólico , Telemetría , TranscriptomaRESUMEN
Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.
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Plaquetas , Células Endoteliales , Vesículas Extracelulares , Vasos Linfáticos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiología , Vasos Linfáticos/metabolismo , Vasos Linfáticos/fisiología , Animales , Células Endoteliales/fisiología , Células Endoteliales/metabolismo , Plaquetas/metabolismo , Plaquetas/fisiología , Ratones , Humanos , Ratones Endogámicos C57BL , Masculino , FemeninoRESUMEN
BACKGROUND: STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH). METHODS: Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2'-deoxyuridine (EdU) assay, and intracellular Ca2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH. RESULTS: Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P<0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1. Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G (STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development. CONCLUSIONS: The potential clinical implications of the 3 noncoding variants of STIM1, rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s).
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Arteria Pulmonar , Molécula de Interacción Estromal 1 , Humanos , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Masculino , Femenino , Estudios de Casos y Controles , Persona de Mediana Edad , Adulto , Proteínas de Neoplasias/genética , Miocitos del Músculo Liso/metabolismo , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Predisposición Genética a la Enfermedad , Músculo Liso Vascular/metabolismo , Regulación de la Expresión Génica , Proliferación Celular/genética , Polimorfismo de Nucleótido SimpleRESUMEN
Lean patients with NAFLD may develop cardiac complications independently of pre-existent metabolic disruptions and comorbidities. To address the underlying mechanisms independent of the development of obesity, we used a murine model of hepatic mitochondrial deficiency. The liver-heart axis was studied as these mice develop microvesicular steatosis without obesity. Our results unveil a sex-dependent phenotypic remodeling beyond liver damage. Males, more than females, show fasting hypoglycemia and increased insulin sensitivity. They exhibit diastolic dysfunction, remodeling of the circulating lipoproteins and cardiac lipidome. Conversely, females do not manifest cardiac dysfunction but exhibit cardiometabolic impairments supported by impaired mitochondrial integrity and ß-oxidation, remodeling of circulating lipoproteins and intracardiac accumulation of deleterious triglycerides. This study underscores metabolic defects in the liver resulting in significant sex-dependent cardiac abnormalities independent of obesity. This experimental model may prove useful to better understand the sex-related variability, notably in the heart, involved in the progression of lean-NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Humanos , Masculino , Femenino , Animales , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Caracteres Sexuales , Modelos Animales de Enfermedad , Obesidad/metabolismo , LipoproteínasRESUMEN
BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Subtilisina/metabolismo , Triglicéridos/metabolismo , Hígado/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proproteína Convertasas/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismoRESUMEN
Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.
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Agrobacterium tumefaciens , Proteínas Bacterianas , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Fimbrias Bacterianas/metabolismo , Membrana Celular/metabolismoRESUMEN
Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid ß-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD-/- mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD-/- mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD-/- mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.
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Acil-CoA Deshidrogenasa de Cadena Larga , Enfermedades Mitocondriales , Ratones , Humanos , Animales , Lactante , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Calcio , Enfermedades Mitocondriales/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados , Arritmias CardíacasRESUMEN
Metabolite genome-wide association studies (mGWAS) have advanced our understanding of the genetic control of metabolite levels. However, interpreting these associations remains challenging due to a lack of tools to annotate gene-metabolite pairs beyond the use of conservative statistical significance threshold. Here, we introduce the shortest reactional distance (SRD) metric, drawing from the comprehensive KEGG database, to enhance the biological interpretation of mGWAS results. We applied this approach to three independent mGWAS, including a case study on sickle cell disease patients. Our analysis reveals an enrichment of small SRD values in reported mGWAS pairs, with SRD values significantly correlating with mGWAS p values, even beyond the standard conservative thresholds. We demonstrate the utility of SRD annotation in identifying potential false negatives and inaccuracies within current metabolic pathway databases. Our findings highlight the SRD metric as an objective, quantitative and easy-to-compute annotation for gene-metabolite pairs, suitable to integrate statistical evidence to biological networks.