Nuevos marcadores trombogénicos en fibrilación auricularimplicación de los microrna en la regulación del componente celular e inflamatorio de la trombosis

Zusammenfassung

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.