Nuevos marcadores trombogénicos en fibrilación auricular: implicación de los microrna en la regulación del componente celular e inflamatorio de la trombosis
- Autores: Ana Belén Arroyo Rodríguez
- Directores de la Tesis: Rocio Gonzalez Conejero Villa (dir. tes.), Constantino Martínez Gómez (dir. tes.)
- Lectura: En la Universidad de Murcia ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Victoriano Francisco Mulero Méndez (presid.), Aránzazu Gonzalez Miqueo (secret.), Carlos Fernández Hernando (voc.)
- Programa de doctorado: Programa de Doctorado en Integración y Modulación de Señales en Biomedicina por la Universidad de Murcia
- Materias:
- Enlaces
- Tesis en acceso abierto en: DIGITUM
- Resumen
- español
La fibrilación auricular (FA), la arritmia cardiaca sostenida más común, se asocia con una alta morbi-mortalidad ya que aumenta el riesgo de sufrir eventos trombóticos. En los últimos años, la etiopatogenia de la FA se ha reconsiderado estableciéndose un vínculo biológico sólido entre trombogénesis e inflamación. La propia arritmia y la coexistencia de factores de riesgo cardiovascular van a favorecer la ocurrencia de eventos cardiovasculares adversos (ECA), siendo la rotura de placas ateroscleróticas el factor más frecuente. Las escala de riesgo clínico CHA2DS2-VASc es la más utilizada para estratificar el riesgo cardiovascular y facilitar la toma de decisiones sobre el tratamiento anticoagulante, aunque presenta una capacidad modesta para predecir los eventos tromboembólicos. Por ello, la búsqueda de nuevos biomarcadores resulta de gran utilidad para mejorar el conocimiento sobre la fisiopatología de la FA y ayudar a refinar la estratificación del riesgo en estos pacientes. En este sentido, los miRNA han surgido como actores críticos de la biología cardiovascular. En particular, miR-146a es un importante regulador negativo de respuestas inmunes e inflamatorias. Además, se han identificado miR-SNP funcionales que modulan los niveles de este miRNA y se asocian con patologías inflamatorias. Recientemente, las trampas extracelulares de neutrófilos (NET) también se han asociado con varias enfermedades inmunes e inflamatorias, incluyendo aterosclerosis y aterotrombosis. De hecho, la perpetuación de NET contribuye al daño tisular y a un estado protrombótico, constituyendo un vínculo entre inflamación y coagulación. En esta Tesis se ha estudiado la implicación de miR-146a y NET en el riesgo trombótico de pacientes con FA así como su potencial uso como biomarcadores de riesgo cardiovascular en esta patología. El trabajo se ha organizado en cuatro capítulos estructurados según los objetivos que se plantean a continuación.
Objetivos: (1) Evaluar el papel pronóstico y el efecto biológico de miR-SNP funcionales de MIR146A en el desarrollo de ECA en FA (Capítulo I). (2) Investigar el papel de miR-146a restringido al compartimento hematopoyético en el desarrollo de aterosclerosis como patología promotora de ECA en FA (Capítulo II). (3) Investigar el papel de miR-146a en la formación de NET y en el desarrollo de trombosis arterial (Capítulo III). (4) Evaluar el papel pronóstico de los NET en el desarrollo de ECA en FA y su relación con el miR-SNP funcional de MIR146A rs2431697 (Capítulo IV).
Material y métodos: Para lograr estos objetivos se utilizaron muestras biológicas de controles y pacientes con FA. Adicionalmente, se utilizaron varios modelos experimentales, incluyendo modelos murinos de sepsis, aterosclerosis y trombosis arterial, así como cultivos primarios de células humanas y de ratón. También se emplearon diversas técnicas de biología celular (citometría de flujo, inmunohistoquímica, inmunofluorescencia, microscopía intravital etc.), técnicas bioquímicas (western blot, ELISA, ensayos fluorimétricos y colorimétricos, etc.) y técnicas de biología molecular (extracción de ácidos nucleicos, genotipado y RT-qPCR). Los resultados obtenidos se analizaron utilizando en cada caso las pruebas estadísticas más apropiadas.
Resultados y conclusiones: Los miR-SNP de MIR146A no se asociaron con el desarrollo de FA. Rs2910164 tampoco se relacionó con ECA, sin embargo el genotipo rs2431697 TT fue un nuevo predictor de riesgo de ECA. Además, los estudios funcionales in vitro en monocitos TT, con niveles más bajos de miR-146a, mostraron un estado pro-inflamatorio mayor que aquellos monocitos CC (Capítulo I). Por otro lado, la deficiencia de miR-146a en células hematopoyéticas moduló los niveles de colesterol en plasma y la expresión de sus dianas en la pared arterial de ratones Ldlr-/- alimentados con dieta grasa pero no aceleró el desarrollo de aterosclerosis (Capítulo II). Además, miR-146a reguló la formación de NET in vitro e in vivo en dos modelos murinos de inflamación (sepsis y aterosclerosis). La deficiencia de miR-146a provocó un mayor grado de trombosis arterial en un modelo murino de daño endotelial inducido con FeCl3 en carótidas (Capítulo III). También demostramos que los niveles plasmáticos de elastasa de neutrófilos, pero no de DNA libre, proporcionan nueva información pronóstica sobre mortalidad y ECA en FA. Finalmente, la variante funcional rs2431697 T, asociada con niveles más bajos de miR-146a, promovió una mayor formación de NET en neutrófilos humanos estimulados in vitro. De acuerdo con estos datos, los pacientes portadores de la variante T para rs2431697 tuvieron los niveles más altos de elastasa de neutrófilos en plasma. Todos estos resultados demuestran por primera vez la implicación de un miRNA en la formación de NET y el riesgo de ECA en FA (Capítulo IV).
En conclusión, esta Tesis responde a la hipótesis de partida y aporta nueva información sobre el riesgo trombótico en FA. Además, proporciona información útil para mejorar la estratificación de riesgo trombótico en pacientes anticoagulados con FA.
- English
Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with high morbidity and mortality, mainly as a consequence of an increased risk of thrombotic events. In recent years, the etiopathogenesis of AF has been reconsidered, establishing a solid biological link between thrombogenesis and inflammation. Arrhythmia itself and the coexistence of other cardiovascular risk factors contribute to the development of adverse cardiovascular events (ACE), being atherosclerotic plaque rupture one of the most frequent triggers for ACE. The CHA2DS2-VASc risk score is the most widely used to stratify cardiovascular risk and facilitate decision-making on anticoagulant therapy, although it has a modest ability to predict thromboembolic events. Therefore, the search for new biomarkers is very important to improve both the knowledge about AF pathophysiology and the stratification risk for these patients. In this framework, miRNAs have emerged as critical players in cardiovascular biology. In particular, miR-146a is an important negative regulator in innate and inflammatory immune responses. Moreover, functional miR-SNPs modulate the levels of this miRNA and are associated with inflammatory pathologies. Recently, neutrophil extracellular traps (NET) have also been associated with several immune and inflammatory diseases, including atherosclerosis and atherothrombosis. Indeed, NET perpetuation contributes to tissue damage and prothrombotic state and represents an important link between inflammation and coagulation. In this Thesis, we have studied the implication of miR-146a and NET formation in the thrombotic risk in AF patients, as well as their potential use as biomarkers of cardiovascular risk in these patients. This study is organized into four chapters according to the objectives set out below.
Objectives: (1) to evaluate the prognostic role and biological effect of functional MIR146A miR-SNPs in the development of ACE in AF (Chapter I). (2) To evaluate the role of miR-146a restricted to the hematopoietic compartment in atherosclerosis as a major comorbidity promoting ACE in AF (Chapter II). (3) To investigate the role of miR-146a in NET formation and arterial thrombosis (Chapter III). (4) To evaluate the prognostic role of NET in the occurrence of ACE in AF and the relation with the functional miR-SNP of MIR146A rs2431697 (Chapter IV).
Materials and methods: To achieve these objectives we obtained biological samples from healthy subjects and AF patients. Additionally, we used different experimental models, i.e mouse models of sepsis, atherosclerosis and arterial thrombosis, as well as human and mouse primary cell culture. We also employed several cell biology techniques (flow cytometry, immunohistochemistry, immunofluorescence, intravital microscopy, etc.), biochemistry techniques (western blot, ELISA, fluorimetric and colorimetric assays, etc.) and molecular biology techniques (nucleic acid extraction, genotyping, and RT-qPCR). The results were subjected to the most appropriate statistical tests.
Results and conclusions: MIR146A miR-SNPs were not associated with AF development and rs2910164 was also unrelated to ACE. However, rs2431697 TT genotype was a new predictor of ACE in AF. In addition, functional studies showed a higher pro-inflammatory state in TT monocytes, associated with lower levels of miR-146a, than in those with CC genotype (Chapter I). Although miR-146a deficiency exclusively affecting the hematopoietic cells modulates cholesterol levels in plasma and the expression of its targets in the artery wall of fat-fed Ldlr-/- mice, our results showed that it does not accelerate atherosclerosis (Chapter II). In addition miR-146a regulates NET formation in vitro and in vivo in two inflammatory murine models (sepsis and atherosclerosis). Moreover, in response to FeCl3-induced vascular injury, miR-146a deficient mice accelerated thrombus formation in carotid arteries (Chapter III). We also reported that neutrophil elastase (NE) plasma levels, but not cell free DNA, are prognostic markers of mortality and ACE in anticoagulated patients with AF. Finally, we showed that functional rs2431697 T variant, associated with lower levels of miR-146a, promotes a higher NET formation in vitro in human neutrophils. Moreover, AF patients with rs2431697 T variant were associated with higher levels of NE. All these results support for the first time the involvement of a miRNA in NET formation and ACE risk in AF (Chapter IV).
In conclusion, this work responds to the initial hypothesis and provides new information about thrombotic risk in AF. Therefore our data could be useful to improve the thrombotic risk stratification in anticoagulated AF patients.
SUMMARY Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with high morbidity and mortality, mainly as a consequence of an increased risk of thrombotic events. In recent years, the etiopathogenesis of AF has been reconsidered, establishing a solid biological link between thrombogenesis and inflammation. Arrhythmia itself and the coexistence of other cardiovascular risk factors contribute to the development of adverse cardiovascular events (ACE), being atherosclerotic plaque rupture one of the most frequent triggers for ACE. The CHA2DS2-VASc risk score is the most widely used to stratify cardiovascular risk and facilitate decision-making on anticoagulant therapy, although it has a modest ability to predict thromboembolic events. Therefore, the search for new biomarkers is very important to improve both the knowledge about AF pathophysiology and the stratification risk for these patients. In this framework, miRNAs have emerged as critical players in cardiovascular biology. In particular, miR-146a is an important negative regulator in innate and inflammatory immune responses. Moreover, functional miR-SNPs modulate the levels of this miRNA and are associated with inflammatory pathologies. Recently, neutrophil extracellular traps (NET) have also been associated with several immune and inflammatory diseases, including atherosclerosis and atherothrombosis. Indeed, NET perpetuation contributes to tissue damage and prothrombotic state and represents an important link between inflammation and coagulation. In this Thesis, we have studied the implication of miR-146a and NET formation in the thrombotic risk in AF patients, as well as their potential use as biomarkers of cardiovascular risk in these patients. This study is organized into four chapters according to the objectives set out below.
Objectives: (1) to evaluate the prognostic role and biological effect of functional MIR146A miR-SNPs in the development of ACE in AF (Chapter I). (2) To evaluate the role of miR-146a restricted to the hematopoietic compartment in atherosclerosis as a major comorbidity promoting ACE in AF (Chapter II). (3) To investigate the role of miR-146a in NET formation and arterial thrombosis (Chapter III). (4) To evaluate the prognostic role of NET in the occurrence of ACE in AF and the relation with the functional miR-SNP of MIR146A rs2431697 (Chapter IV).
Materials and methods: To achieve these objectives we obtained biological samples from healthy subjects and AF patients. Additionally, we used different experimental models, i.e mouse models of sepsis, atherosclerosis and arterial thrombosis, as well as human and mouse primary cell culture. We also employed several cell biology techniques (flow cytometry, immunohistochemistry, immunofluorescence, intravital microscopy, etc.), biochemistry techniques (western blot, ELISA, fluorimetric and colorimetric assays, etc.) and molecular biology techniques (nucleic acid extraction, genotyping, and RT-qPCR). The results were subjected to the most appropriate statistical tests.
Results and conclusions: MIR146A miR-SNPs were not associated with AF development and rs2910164 was also unrelated to ACE. However, rs2431697 TT genotype was a new predictor of ACE in AF. In addition, functional studies showed a higher pro-inflammatory state in TT monocytes, associated with lower levels of miR-146a, than in those with CC genotype (Chapter I). Although miR-146a deficiency exclusively affecting the hematopoietic cells modulates cholesterol levels in plasma and the expression of its targets in the artery wall of fat-fed Ldlr-/- mice, our results showed that it does not accelerate atherosclerosis (Chapter II). In addition miR-146a regulates NET formation in vitro and in vivo in two inflammatory murine models (sepsis and atherosclerosis). Moreover, in response to FeCl3-induced vascular injury, miR-146a deficient mice accelerated thrombus formation in carotid arteries (Chapter III). We also reported that neutrophil elastase (NE) plasma levels, but not cell free DNA, are prognostic markers of mortality and ACE in anticoagulated patients with AF. Finally, we showed that functional rs2431697 T variant, associated with lower levels of miR-146a, promotes a higher NET formation in vitro in human neutrophils. Moreover, AF patients with rs2431697 T variant were associated with higher levels of NE. All these results support for the first time the involvement of a miRNA in NET formation and ACE risk in AF (Chapter IV).
In conclusion, this work responds to the initial hypothesis and provides new information about thrombotic risk in AF. Therefore our data could be useful to improve the thrombotic risk stratification in anticoagulated AF patients.
- español
