Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.
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ABSTRACT:
SUBMITTER: Gaul DS
PROVIDER: S-EPMC6014146 | biostudies-literature | 2018 Jul
REPOSITORIES: biostudies-literature
Publications
Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.
Gaul Daniel S DS Weber Julien J van Tits Lambertus J LJ Sluka Susanna S Pasterk Lisa L Reiner Martin F MF Calatayud Natacha N Lohmann Christine C Klingenberg Roland R Pahla Jürgen J Vdovenko Daria D Tanner Felix C FC Camici Giovanni G GG Eriksson Urs U Auwerx Johan J Mach François F Windecker Stephan S Rodondi Nicolas N Lüscher Thomas F TF Winnik Stephan S Matter Christian M CM
Cardiovascular research 20180701 8
<h4>Aims</h4>Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).<h4>Methods and results</h4 ...[more]
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Project description:Studies of patients with COVID-19 have demonstrated markedly dysregulated coagulation and a high risk of morbid arterial and venous thrombotic events. Elevated levels of blood neutrophils and neutrophil extracellular traps (NETs) have recently been described in patients with COVID-19. However, their potential role in COVID-19-associated thrombosis remains incompletely understood. In order to elucidate the potential role of hyperactive neutrophils and NET release in COVID-19-associated thrombosis, we conducted a case-control study of patients hospitalized with COVID-19 who developed thrombosis, as compared with gender- and age-matched COVID-19 patients without clinical thrombosis. We found that remnants of NETs (cell-free DNA, myeloperoxidase-DNA complexes, and citrullinated histone H3) and neutrophil-derived S100A8/A9 (calprotectin) in patient sera were associated with higher risk of morbid thrombotic events in spite of prophylactic anticoagulation. These observations underscore the need for urgent investigation into the potential relationship between NETs and unrelenting thrombosis in COVID-19, as well as novel approaches for thrombosis prevention.
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Project description:Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Neutrophils are a component of the innate immune system which protect against pathogen invasion; however, the contribution of neutrophils to cardiovascular disease has been underestimated, despite infiltration of leukocyte subsets being a known driving force of atherosclerosis and thrombosis. In addition to their function as phagocytes, neutrophils can release their extracellular chromatin, nuclear protein, and serine proteases to form net-like fiber structures, termed neutrophil extracellular traps (NETs). NETs can entrap pathogens, induce endothelial activation, and trigger coagulation, and have been detected in atherosclerotic and thrombotic lesions in both humans and mice. Moreover, NETs can induce endothelial dysfunction and trigger proinflammatory immune responses. Overall, current data indicate that NETs are not only present in plaques and thrombi but also have causative roles in triggering formation of atherosclerotic plaques and venous thrombi. This review is focused on published findings regarding NET-associated endothelial dysfunction during atherosclerosis, atherothrombosis, and venous thrombosis pathogenesis. The NET structure is a novel discovery that will find its appropriate place in our new understanding of cardiovascular disease. In addition, NETs have high potential to be further explored toward much better treatment of atherosclerosis and venous thromboembolism in clinic.
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Project description:BackgroundNeutrophil extracellular traps (NETs) contribute to the creation of a coagulation state in various diseases. Currently, it is not clear whether NETs are present in the thrombi and plasma of patients with cerebral venous sinus thrombosis (CVST). This study aimed to investigate the presence of NETs in thrombi and blood samples from CVST patients and the procoagulant activity (PCA) of NETs during the progression of CVST.ResultsThrombi obtained from CVST patients undergoing thrombectomy were examined by immunochemistry using neutrophil elastase (NE), CD66b and citrullinated histone H3(citH3). The presence of NET markers in samples from 37 CVST patients and 32 healthy people was evaluated by ELISA. NET-producing neutrophils and neutrophil-platelet (PLT) aggregates were examined in samples obtained from CVST patients and healthy people by flow cytometry. The TAT complex in plasma sample from each group was detected by ELISA to evaluate the procoagulant activity of NETs in CVST patients. Neutrophils from healthy subjects were treated with PLT-rich plasma in the presence of anti-PF4 antibodies or an autophagy inhibitor and analyzed by flow cytometry and confocal microscopy. After treatment with NETs, the expression of von Willebrand factor (VWF), tissue factor (TF) and CD31 in human brain microvascular endothelial cells (HBMECs) was measured by confocal microscopy and western blotting. Our results showed that NETs were abundant in the plasma and thrombi from CVST patients. Platelet factor 4 (PF4) from CVST PLTs induced NET generation through autophagy. NETs could induce PCA by modulating TF and phosphatidylserine (PS) in CVST. NETs also disrupted the endothelial barrier and transformed ECs into a procoagulant phenotype to exacerbate thrombogenicity.ConclusionsNET generation was mediated by PF4 from PLTs through autophagy and contribute to thrombosis in CVST patients.
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Project description:ObjectivesThe aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA.MethodsWe compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model.ResultsNeutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex.ConclusionWe demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA.
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Project description:Deep vein thrombosis (DVT) is linked to local inflammation. A role for both neutrophil extracellular traps (NETs) and the assembly of inflammasomes (leading to caspase-1-dependent interleukin-1β activation) in the development of DVT was recently suggested. However, no link between these 2 processes in the setting of thrombosis has been investigated. Here, we demonstrate that stimulation of neutrophils induced simultaneous formation of NETs and active caspase-1. Caspase-1 was largely associated with NETs, suggesting that secreted active caspase-1 requires NETs as an adhesive surface. NETs and their components, histones, promoted robust caspase-1 activation in platelets with the strongest effect exerted by histones 3/4. Murine DVT thrombi contained active caspase-1, which peaked at 6 hours when compared with 48-hour thrombi. Platelets constituted more than one-half of cells containing active caspase-1 in dissociated thrombi. Using intravital microscopy, we identified colocalized NETs and caspase-1 as well as platelet recruitment at the site of thrombosis. Pharmacological inhibition of caspase-1 strongly reduced DVT in mice, and thrombi that still formed contained no citrullinated histone 3, a marker of NETs. Taken together, these data demonstrate a cross-talk between NETs and inflammasomes both in vitro and in the DVT setting. This may be an important mechanism supporting thrombosis in veins.
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Project description:BackgroundThrombotic events cannot be completely prevented by antithrombotics, implicating a therapeutic gap due to inflammation, a not yet sufficiently addressed mechanism. Neutrophil extracellular traps (NETs) are an essential interface between inflammation and thrombosis, but exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood.Methods and resultsWe found that the plasma concentrations of C5a were higher in patients with ST-elevation myocardial infarction (STEMI) than in patients with angina and higher in mice with left common carotid artery (LCCA) thrombosis induced by FeCl3 than in control mice. We observed that the thrombus area and weight were decreased and that NET formation in the thrombi was reduced in the group treated with the selective C5aR1 receptor inhibitor PMX53 compared with the NaCl group. In vitro, NETosis was observed when C5a was added to neutrophil cultures, and this effect was reversed by PMX53. In addition, our data showed that C5a increased the production of mitochondrial reactive oxygen species (ROS) and that the promotion of NET formation by C5a was mitochondrial ROS (Mito-ROS) dependent. Furthermore, we found that C5a induced the production of Mito-ROS by inhibiting mitochondrial STAT3 activity.ConclusionsBy inhibiting mitochondrial STAT3 to elicit Mito-ROS generation, C5a triggers the generation of NETs to promote the development of arterial thrombosis. Hence, our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis.
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Project description:ObjectiveAntiphospholipid syndrome (APS) is a leading acquired cause of thrombotic events. Although antiphospholipid antibodies have been shown to promote thrombosis in mice, the role of neutrophils has not been explicitly studied. The aim of this study was to characterize neutrophils in the context of a new model of antiphospholipid antibody-mediated venous thrombosis.MethodsMice were administered fractions of IgG obtained from patients with APS. At the same time, blood flow through the inferior vena cava was reduced by induction of stenosis. Resulting thrombi were characterized for size and neutrophil content. Circulating factors and the vessel wall were also assessed.ResultsAs measured by both thrombus weight and thrombosis frequency, mice treated with IgG from patients with APS (APS IgG) demonstrated exaggerated thrombosis as compared with control IgG-treated mice. Thrombi in mice treated with APS IgG were enriched for citrullinated histone H3 (a marker of neutrophil extracellular traps [NETs]). APS IgG-treated mice also demonstrated elevated levels of circulating cell-free DNA and human IgG bound to the neutrophil surface. In contrast, circulating neutrophil numbers and markers of vessel wall activation were not appreciably different between APS IgG-treated mice and control mice. Treatment with either DNase (which dissolves NETs) or a neutrophil-depleting antibody reduced thrombosis in APS IgG-treated mice to the level in control mice.ConclusionThese data support a mechanism whereby circulating neutrophils are primed by antiphospholipid antibodies to accelerate thrombosis. This line of investigation suggests new, immunomodulatory approaches for the treatment of APS.
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