RESUMO
AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.
Assuntos
Infarto do Miocárdio com Supradesnível do Segmento ST , Trombose , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Trombose/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD+-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. APPROACH AND RESULTS: Using a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6-/- mice as compared to Sirt6fl/fl controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. CONCLUSIONS: Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.
Assuntos
Sirtuínas , Trombose , Animais , Humanos , Masculino , Camundongos , Células Cultivadas , Ciclo-Oxigenase 2 , Células Endoteliais , NF-kappa B , Inibidores de Poli(ADP-Ribose) Polimerases , Sirtuínas/genética , Trombose/genética , Fator de Necrose Tumoral alfa , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
AIMS: Epidemiologic evidence links ischemic stroke to age, yet the mechanisms that underlie the specific and independent effects of age on stroke remain elusive, impeding the development of targeted treatments. This study tested the hypothesis that age directly aggravates stroke outcomes and proposes inflamm-aging as a mediator and potential therapeutic target. METHODS: 3 months- (young) and 18-20 months-old (old) mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 minutes followed by 48 hours of reperfusion. Old animals received weekly treatment with the TNF-α neutralizing antibody adalimumab over 4 weeks before tMCAO in a separate set of experiments. Plasma levels of TNF- α were assessed in patients with ischemic stroke and correlated with age and outcome. RESULTS: Old mice displayed larger stroke size than young ones with increased neuromotor deficit. Immunohistochemical analysis revealed impairment of the blood-brain barrier in old mice, i.e. increased post-stroke degradation of endothelial tight junctions and expression of tight junctions-digesting and neurotoxic matrix metalloproteinases. At baseline, old animals showed a broad modulation of several circulating inflammatory mediators. TNF-α displayed the highest increase in old animals and its inhibition restored the volume of stroke, neuromotor performance, and survival rates of old mice to the levels observed in young ones. Patients with ischemic stroke showed increased TNF-α plasma levels which correlated with worsened short-term neurological outcome as well as with age. CONCLUSIONS: This study identifies TNF-α as a causative contributor to the deleterious effect of aging on stroke and points to inflamm-aging as a mechanism of age-related worsening of stroke outcomes and potential therapeutic target in this context. Thus, this work provides a basis for tailoring novel stroke therapies for the particularly vulnerable elderly population.
Assuntos
Adalimumab/farmacologia , Envelhecimento/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Inibidores do Fator de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Caderinas/metabolismo , Feminino , Humanos , Interleucina-1beta/metabolismo , AVC Isquêmico/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Traumatismo por Reperfusão/metabolismo , Proteínas de Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: Aging is an established risk factor for stroke; genes regulating longevity are implicated in the pathogenesis of ischaemic stroke where to date, therapeutic options remain limited. The blood-brain barrier (BBB) is crucially involved in ischaemia/reperfusion (I/R) brain injury thus representing an attractive target for developing novel therapeutic agents. Given the role of endothelial cells in the BBB, we hypothesized that the endothelial-specific expression of the recently described longevity gene SIRT6 may exhibit protective properties in stroke. METHODS AND RESULTS: SIRT6 endothelial expression was reduced following stroke. Endothelial-specific Sirt6 knockout (eSirt6-/-) mice, as well as animals in which Sirt6 overexpression was post-ischaemically induced, underwent transient middle cerebral artery occlusion (tMCAO). eSirt6-/- animals displayed increased infarct volumes, mortality, and neurological deficit after tMCAO, as compared to control littermates. Conversely, post-ischaemic Sirt6 overexpression decreased infarct size and neurological deficit. Analysis of ischaemic brain sections revealed increased BBB damage and endothelial expression of cleaved caspase-3 in eSIRT6-/- mice as compared to controls. In primary human brain microvascular endothelial cells (HBMVECs), hypoxia/reoxygenation (H/R) reduced SIRT6 expression and SIRT6 silencing impaired the barrier function (transendothelial resistance) similar to what was observed in mice exposed to I/R. Further, SIRT6-silenced HBMVECs exposed to H/R showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the survival pathway Akt. In ischaemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement as assessed by NIHSS scale and correlated with stroke outcome. CONCLUSION: Endothelial SIRT6 exerts a protective role in ischaemic stroke by blunting I/R-mediated BBB damage and thus, it may represent an interesting novel therapeutic target to be explored in future clinical investigation.
Assuntos
Isquemia Encefálica , Sirtuínas , Acidente Vascular Cerebral , Animais , Barreira Hematoencefálica , Células Endoteliais , Humanos , Infarto da Artéria Cerebral Média , Camundongos , Camundongos Endogâmicos C57BL , Sirtuínas/genéticaRESUMO
BACKGROUND: Endothelial cells regulate the formation of blood clots; thus, genes selectively expressed in these cells could primarily determine thrombus formation. Apold1 (apolipoprotein L domain containing 1) is a gene expressed by endothelial cells; whether Apold1 directly contributes to arterial thrombosis has not yet been investigated. Here, we assessed the effect of Apold1 deletion on arterial thrombus formation using an in vivo model of carotid thrombosis induced by photochemical injury. MATERIAL AND METHODS: Apold1 knockout (Apold1-/- ) mice and wild-type (WT) littermates underwent carotid thrombosis induced by photochemical injury, and time to occlusion was recorded. Tissue factor (TF) activity and activation of mitogen-activated protein kinases (MAPKs) and phosphatidyl-inositol-3 kinase (PI3K)/Akt pathways were analysed by colorimetric assay and Western blotting in both Apold1-/- and WT mice. Finally, platelet reactivity was assessed using light transmission aggregometry. RESULTS: After photochemical injury, Apold1-/- mice exhibited shorter time to occlusion as compared to WT mice. Moreover, TF activity was increased in carotid arteries of Apold1-/- when compared to WT mice. Underlying mechanistic markers such as TF mRNA and MAPKs activation were unaffected in Apold1-/- mice. In contrast, phosphorylation of Akt was reduced in Apold1-/- as compared to WT mice. Additionally, Apold1-/- mice displayed increased platelet reactivity to stimulation with collagen compared with WT animals. CONCLUSIONS: Deficiency of Apold1 results in a prothrombotic phenotype, accompanied by increased vascular TF activity, decreased PI3K/Akt activation and increased platelet reactivity. These findings suggest Apold1 as an interesting new therapeutic target in the context of arterial thrombosis.
Assuntos
Trombose das Artérias Carótidas/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Plaquetária/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tromboplastina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Células Endoteliais/metabolismo , Corantes Fluorescentes , Proteínas Imediatamente Precoces/genética , Fotocoagulação a Laser , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Processos Fotoquímicos , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Rosa Bengala , Transdução de Sinais , Tromboplastina/genéticaRESUMO
Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2 Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-ß cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas , Ácido alfa-Linolênico , Animais , Plaquetas , Análise por Conglomerados , Tomografia com Microscopia Eletrônica , Humanos , Ácido alfa-Linolênico/farmacologia , Fator de von WillebrandRESUMO
Background and Purpose- Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD-a member of the AP-1 (activated protein-1) family of transcription factors-was recently shown to regulate inflammation by targeting IL (interleukin)-1ß synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods- WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1ß was investigated by treating JunD siRNA mice with an anti-IL-1ß monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results- In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1ß levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti-IL-1ß antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions- JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1ß.
Assuntos
Lesões Encefálicas/metabolismo , Interleucina-1beta/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Regulação da Expressão Gênica , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-jun/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Aims: The CANTOS trial underscored the efficacy of selective antibody-based interleukin (IL)-1ß inhibition with Canakinumab in secondary prevention of cardiovascular events. Despite the success of the trial, incidence of stroke was not reduced likely due to the low number of events and the relatively young age of patients enrolled. Given the established role of IL-1ß in stroke, we tested the efficacy of the murine Canakinumab-equivalent antibody in a mouse model of ischaemic stroke. To mimic the clinical scenario of modern stroke management, IL-1ß inhibition was performed post-ischaemically upon reperfusion as it would be the case in patients presenting to the emergency room and eligible for thrombolytic therapy. Methods and results: Transient middle cerebral artery occlusion (tMCAO) was performed in wild type mice; upon reperfusion, mice were randomly allocated to anti-IL-1ß antibody or vehicle treatment. Following tMCAO, cerebral IL-1ß levels, unlike tumour necrosis factor-α, were increased underscoring a role for this cytokine. Post-ischaemic treatment with IL-1ß antibody reduced infarct size, cerebral oedema and improved neurological performance as assessed by 2,3,5-triphenyltetrazolium chloride staining, Bederson and RotaRod tests. Antibody-treated animals also exhibited a reduced neutrophil and matrix metalloproteinase (MMP)-2 but not MMP-9, activity in ipsilateral hemispheres as compared to vehicle-treated mice. Noteworthy, tMCAO associated vascular endothelial-cadherin reduction was blunted in IL-1ß antibody-treated mice compared to vehicle-treated, likely providing the mechanistic explanation for the improved outcome. Conclusion: Our data for the first time demonstrate the efficacy of selective post-ischaemic IL-1ß blockade in improving outcome following experimental ischaemia/reperfusion brain injury in the mouse and encourage further focused clinical studies assessing the potential of the approved IL-1ß antibody Canakinumab, as an adjuvant therapy to thrombolysis in acute ischaemic stroke patients.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Infarto da Artéria Cerebral Média/prevenção & controle , Interleucina-1beta/antagonistas & inibidores , Acidente Vascular Cerebral/prevenção & controle , Animais , Anticorpos Monoclonais Humanizados , Encéfalo/metabolismo , Caderinas/metabolismo , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Elastase de Leucócito/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Distribuição Aleatória , Prevenção SecundáriaRESUMO
AIMS: Oral anticoagulation is considered standard therapy for stroke prevention in atrial fibrillation (AF). Endocardial activation triggers expression of pro-thrombotic mediators including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), and contributes to thrombus formation in the left atrial appendage (LAA) of AF patients. Recently, pleiotropic effects of specific P2Y12 receptor antagonists were demonstrated; however, whether these drugs possess antithrombotic effects on LAA endocardial cells currently remains unknown. METHODS AND RESULTS: LAA were obtained from 14 patients with known AF undergoing elective cardiac surgery including LAA removal at the University Hospital Zurich. LAA endocardial cells were isolated and pre-incubated with ticagrelor (10-7, 10-6, 10-5M) or clopidogrel active metabolite (CAM) (1.5 × 10-8, 1.5 × 10-7, 1.5 × 10-6 M) before stimulation with tumour necrosis factor-alpha (TNF-α) (10 ng/mL). Finally, TF and PAI-1 expression and activity were analysed. Ticagrelor, unlike CAM, concentration dependently decreased TNF-α-induced TF expression and TF activity in LAA endocardial cells. Further, ticagrelor, but not CAM reduced PAI-1 expression and enzyme activity in TNF-α-stimulated LAA endocardial cells. In contrast, TF pathway inhibitor (TFPI) remained unaffected by both dugs. CONCLUSION: Ticagrelor, but not CAM, reduces expression and activity of TF and PAI-1 in LAA endocardial cells isolated from patients with AF, indicating possible local antithrombotic effects. Such pleiotropic properties of ticagrelor may contribute to a reduction in thromboembolic complications in patients with AF.
Assuntos
Adenosina/análogos & derivados , Antitrombinas/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ticlopidina/análogos & derivados , Adenosina/farmacologia , Apêndice Atrial , Fibrilação Atrial , Clopidogrel , Endocárdio/metabolismo , Átrios do Coração , Humanos , Inibidor 1 de Ativador de Plasminogênio/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Tromboplastina/antagonistas & inibidores , Ticagrelor , Ticlopidina/metabolismo , Ticlopidina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Furocumarinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/efeitos da radiação , Raios Ultravioleta , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Colágeno/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos da radiação , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos da radiação , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator de von Willebrand/metabolismoAssuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Interleucina-1alfa/antagonistas & inibidores , AVC Isquêmico/etiologia , AVC Isquêmico/metabolismo , Animais , Infarto Encefálico/etiologia , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Endotelinas/metabolismo , AVC Isquêmico/diagnóstico , AVC Isquêmico/terapia , Masculino , CamundongosRESUMO
Patients often complain of fatigue, sleepiness or tiredness to their treating physician. Somatic causes should not be missed. According to answers in a survey involving experienced Swiss Heads of medicine departments, somatic causes of fatigue are discussed in this article with focus on obstacles and near-missed cases. Diagnostic tools and treatment options, if available, are mentioned.
Assuntos
Distúrbios do Sono por Sonolência Excessiva/etiologia , Fadiga/etiologia , Coleta de Dados , Distúrbios do Sono por Sonolência Excessiva/diagnóstico , Fadiga/diagnóstico , Humanos , SuíçaAssuntos
Aterosclerose , Trombose , Aterosclerose/complicações , Humanos , Sistema de Registros , Trombose/terapiaRESUMO
INTRODUCTION: Arterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3-0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma. MATERIALS AND METHODS: Platelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured. RESULTS: APAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition. CONCLUSIONS: Intravenous APAC targets arterial injury sites to exert local dual antiplatelet and anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, highlighting APAC as a novel antithrombotic to reduce cardiovascular complications.
Assuntos
Trombose das Artérias Carótidas , Trombose , Lesões do Sistema Vascular , Animais , Camundongos , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Anticoagulantes/química , Tromboplastina , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Fibrinolíticos/uso terapêutico , Trombose/etiologia , Trombose das Artérias Carótidas/tratamento farmacológico , Colágeno/farmacologia , Agregação PlaquetáriaRESUMO
AIMS: The prevalence of left ventricular (LV) diastolic and vascular dysfunction increases with age, eventually leading to heart failure with preserved ejection fraction (HFpEF). A preventive strategy is an unmet medical need. We and others reported previously on the beneficial effects of omega-3 fatty acid alpha linolenic acid (ALA) on cardiovascular disorders in animal models and translational studies. We now investigate whether long-term dietary ALA could prevent LV diastolic dysfunction and vascular aging in a murine model. METHODS AND RESULTS: Wild-type C57BL/6â¯J mice were fed a chow or ALA diet for 12â¯months, starting at 6â¯months of age. Here, we show that aged (~18â¯months) mice recapitulate major hallmarks of HFpEF, including LV diastolic dysfunction with preserved ejection fraction, impaired vascular function, cardiac fibrosis, arterial stiffening and inflammation, as well as elevated B-type natriuretic peptide (BNP). Long-term ALA supplementation upregulated the mitochondrial tricarboxylic acid enzyme Idh2 and the antioxidant enzymes SOD1 and Gpx1. It also has been associated with reduced inflammation and ECM remodeling, accompanied by a significant downregulation of fibrosis biomarkers MMP-2 and TGF-ß in both cardiac and vascular tissues obtained from aged mice. Our data exhibited the preventive effects of dietary ALA against LV diastolic dysfunction, impaired vasorelaxation, cardiac fibrosis, inflammation and arterial stiffening in aged mice. CONCLUSIONS: We provide evidence and a simplified mechanistic insight on how long-term ALA supplementation is a successful strategy to prevent the development of age-related diastolic and vascular dysfunction.
Assuntos
Ácidos Graxos Ômega-3 , Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Camundongos , Animais , Ácidos Graxos Ômega-3/farmacologia , Volume Sistólico/fisiologia , Camundongos Endogâmicos C57BL , Disfunção Ventricular Esquerda/prevenção & controle , Envelhecimento , Fibrose , Ácidos Graxos , Inflamação , DietaRESUMO
AIMS: Low-grade inflammation couples dysmetabolic states to insulin resistance and atherosclerotic cardiovascular (CV) disease (ASCVD). Selective sodium-glucose co-transporter 2 (SGLT-2) inhibition by empagliflozin improves clinical outcomes in patients with ASCVD independently of its glucose lowering effects. Yet, its mechanism of action remains largely undetermined. Here, we aimed to test whether empagliflozin affects arterial thrombus formation in baseline (BSL) conditions or low-grade inflammatory states, a systemic milieu shared among patients with ASCVD. METHODS AND RESULTS: Sixteen-week-old C57BL/6 mice were randomly assigned to acute administration of empagliflozin (25 mg/kg body weight) or vehicle, of which a subgroup was pre-treated biweekly over 4 weeks with super-low-dose lipopolysaccharide (LPS; 5 ng/kg body weight), before carotid thrombosis was induced by photochemical injury. The between-group difference in Doppler-flow probe detected time-to-occlusion remained within the predefined equivalence margin (Δ = |10.50|), irrespective of low-grade inflammation (95% confidence interval, -9.82 to 8.85 and -9.20 to 9.69), while glucose dropped by 1.64 and 4.84 mmoL/L, respectively. Ex vivo platelet aggregometry suggested similar activation status, corroborated by unchanged circulating platelet-factor 4 plasma levels. In concert, carotid PAI-1 expression and tissue factor (TF) activity remained unaltered upon SGLT-2 inhibition, and no difference in plasma D-dimer levels was detected, suggesting comparable coagulation cascade activation and fibrinolytic activity. In human aortic endothelial cells pre-treated with LPS, empagliflozin neither changed TF activity nor PAI-1 expression. Accordingly, among patients with established ASCVD or at high CV risk randomized to a daily dose of 10 mg empagliflozin signatures of thrombotic (i.e. TF) and fibrinolytic activity (i.e. PAI-1) remained unchanged, while plasma glucose declined significantly during 3 months of follow-up. CONCLUSION: SGLT-2 inhibition by empagliflozin does not impact experimental arterial thrombus formation, neither under BSL conditions nor during sustained low-grade inflammation, and has no impact on proxies of thrombotic/fibrinolytic activity in patients with ASCVD. The beneficial pleiotropic effects of empagliflozin are likely independent of pathways mediating arterial thrombosis.
Assuntos
Diabetes Mellitus Tipo 2 , Inibidores do Transportador 2 de Sódio-Glicose , Trombose , Humanos , Camundongos , Animais , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais , Transportador 2 de Glucose-Sódio , Lipopolissacarídeos/uso terapêutico , Inibidor 1 de Ativador de Plasminogênio , Camundongos Endogâmicos C57BL , Trombose/induzido quimicamente , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Glucose , Inflamação/tratamento farmacológico , Peso Corporal , Diabetes Mellitus Tipo 2/tratamento farmacológicoRESUMO
AIMS: The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-ß superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. METHODS AND RESULTS: In contrast to endogenous Mstn, myocardial Gdf11 declined during the course of ageing and was particularly reduced following ischaemia/reperfusion (I/R) injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by recombinant GDF11 delivery (0.1â mg/kg body weight over 30 days) prior to myocardial I/R augmented myocardial infarct size in C57BL/6 mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. While intrinsic cardioprotective signalling pathways remained unaffected by high circulating GDF11, targeted transcriptomics and immunomapping studies focusing on GDF11-associated downstream targets revealed attenuated Nkx2-5 expression confined to CD105-expressing cells, with pro-apoptotic activity, as assessed by caspase-3 levels, being particularly pronounced in adjacent cells, suggesting an indirect effect. By harnessing a highly specific and validated liquid chromatography-tandem mass spectrometry-based assay, we show that in prospectively recruited patients with MI circulating GDF11 but not MSTN levels incline with age. Moreover, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event, with circulating GDF11 emerging as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. CONCLUSION: Our data challenge the initially reported heart rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice and humans alike. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of cardioprotective mechanisms and thus poor outcomes of elderly patients following acute MI.
Assuntos
Fatores de Diferenciação de Crescimento , Traumatismos Cardíacos , Infarto do Miocárdio , Idoso , Animais , Humanos , Camundongos , Envelhecimento/metabolismo , Proteínas Morfogenéticas Ósseas , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Coração , Traumatismos Cardíacos/complicações , Traumatismos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismoRESUMO
Background Previous randomized control trials showed mixed results concerning the effect of omega-3 fatty acids (n-3 FAs) on atrial fibrillation (AF). The associations of n-3 FA blood levels with heart rhythm in patients with established AF are unknown. The goal of this study was to assess the associations of total and individual n-3 FA blood levels with AF type (paroxysmal versus nonparoxysmal), heart rate (HR), and HR variability in patients with AF. Methods and Results Total n-3 FAs, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, and alpha-linolenic acid blood levels were determined in 1969 patients with known AF from the SWISS-AF (Swiss Atrial Fibrillation cohort). Individual and total n-3 FAs were correlated with type of AF, HR, and HR variability using standard logistic and linear regression, adjusted for potential confounders. Only a mild association with nonparoxysmal AF was found with total n-3 FA (odds ratio [OR], 0.97 [95% CI, 0.89-1.05]) and docosahexaenoic acid (OR, 0.93 [95% CI, 0.82-1.06]), whereas other individual n-3 FAs showed no association with nonparoxysmal AF. Higher total n-3 FAs (estimate 0.99 [95% CI, 0.98-1.00]) and higher docosahexaenoic acid (0.99 [95% CI, 0.97-1.00]) tended to be associated with slower HR in multivariate analysis. Docosapentaenoic acid was associated with a lower HR variability triangular index (0.94 [95% CI, 0.89-0.99]). Conclusions We found no strong evidence for an association of n-3 FA blood levels with AF type, but higher total n-3 FA levels and docosahexaenoic acid might correlate with lower HR, and docosapentaenoic acid with a lower HR variability triangular index.
Assuntos
Fibrilação Atrial , Ácidos Graxos Ômega-3 , Humanos , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/tratamento farmacológico , Ácidos Docosa-Hexaenoicos , Seguimentos , Ácido Eicosapentaenoico , Frequência Cardíaca/fisiologiaRESUMO
BACKGROUND: Sickle cell disease (SCD) is a genetic hemoglobinopathy associated with high morbidity and mortality. The primary cause of hospitalization in SCD is vaso-occlusive crisis (VOC), mediated by alteration of red blood cells, platelets, immune cells and a pro-adhesive endothelium. OBJECTIVES: We investigated the potential therapeutic use of the plant-derived omega-3 alpha-linolenic acid (ALA) in SCD. METHODS: Berkeley mice were fed a low- or high-ALA diet for 4 weeks, followed by analysis of liver fibrosis, endothelial activation, platelet activation and formation of platelet-neutrophils aggregates. Aggregation of platelets over collagen under flow after high-ALA was compared to a blocking P-selectin Fab. RESULTS: Dietary high-ALA was able to reduce the number of sickle cells in blood smear, liver fibrosis, and the expression of adhesion molecules on the endothelium of aorta, lungs, liver and kidneys (VCAM-1, ICAM-1 and vWF). Specific parameters of platelet activation were blunted after high-ALA feeding, notably P-selectin exposure and the formation of neutrophil-platelet aggregates, along with a correspondingly reduced expression of PSGL-1 on neutrophils. By comparison, in vivo treatment of SCD mice with the anti-P-selectin Fab was able to similarly reduce the formation of neutrophil-platelet aggregates, but did not reduce GpIbα shedding nor the activation of the αIIb ß3 integrin in response to thrombin. Both ALA feeding and P-selectin blocking significantly reduced collagen-mediated cell adhesion under flow. CONCLUSIONS: Dietary ALA is able to reduce the pro-inflammatory and pro-thrombotic state occurring in the SCD mouse model and may represent a novel, inexpensive and readily available therapeutic strategy for SCD.