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In viral infection, morbidity and mortality often result from extrahepatic disease manifestations such as vasculitis. We hereby show that human microvascular endothelial cells express viral receptors of the innate immune system which are induced upon ligand engagement. Furthermore, stimulation of endothelial cells with the synthetic analog of viral DNA, poly (dA:dT), human DNA and hepatitis B virus-containing immunoprecipitates from a patient with polyarteritis nodosa induces an inflammatory response including the upregulation of adhesion molecules, which is mediated exclusively by TLR9 and involves an IRF3-dependent pathway. Thus, endothelial cells are able to actively participate in immune mediated vascular inflammation caused by viral infections. Furthermore, we provide evidence for the ability of LL37 to bind and internalize viral or endogenous DNA into non-immune cells. DNA nucleotides internalized by LL37 suppress the production of proinflammatory mediators suggesting a protective effect against direct responses to viral infection or circulating DNA-fragments of endogenous origin.
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Catelicidinas/inmunología , ADN Viral/inmunología , Células Endoteliales/inmunología , Inflamación/inmunología , Microvasos/inmunología , Poli dA-dT/inmunología , Péptidos Catiónicos Antimicrobianos , Catelicidinas/metabolismo , Células Cultivadas , Quimiocinas/inmunología , Quimiocinas/metabolismo , Trampas Extracelulares/metabolismo , Virus de la Hepatitis B/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Inflamación/metabolismo , Inflamación/virología , Factor 3 Regulador del Interferón/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.
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Aspirina/metabolismo , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Sulfuro de Hidrógeno/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Animales , Aspirina/análogos & derivados , Tiempo de Sangría , Plaquetas/metabolismo , AMP Cíclico/metabolismo , Disulfuros/farmacología , Humanos , Técnicas In Vitro , Integrinas/efectos de los fármacos , Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Agregación Plaquetaria/fisiología , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Trombosis/metabolismo , Trombosis/prevención & controlRESUMEN
INTRODUCTION: Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα -induced endothelial inflammation in vivo. METHODS: Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. RESULTS: Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα -induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα -induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα -induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. CONCLUSIONS: The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF α -induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.
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Plaquetas/metabolismo , Endotelio/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Gluconato de Sodio Antimonio/farmacología , Western Blotting , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína Tirosina Fosfatasa no Receptora Tipo 6/antagonistas & inhibidores , PorcinosRESUMEN
INTRODUCTION: Catheter ablation is the treatment of choice for recurrent focal atrial tachycardia (FAT) as medical therapy is limited. Routinely, a three-dimensional mapping system is used. Whether or not optimized signal detection does improve ablation success rates has not yet been investigated. This retrospective cohort study compared ablation procedures using an ultra-high-density mapping system (UHDM, Rhythmia, Boston Scientific) with improved signal detection and automatic annotation with procedures using a conventional electroanatomic mapping system (CEAM, Biosense Webster, CARTO). METHODS: All patients undergoing ablation for FAT using UHDM or CEAM from April 2015 to August 2018 were included. Endpoints comprised procedural parameters, acute success as well as freedom from arrhythmia 12 months after ablation. RESULTS: A total of 70 patients underwent ablation (48 with UHDM, 22 with CEAM). No significant differences were noted for parameters like procedural and radiation duration, area dose, and RF applications. Acute success was significantly higher in the UHDM cohort (89.6% vs. 68.2%, p = .03). Nevertheless, arrhythmia freedom 12 months after ablation was almost identical (56.8% vs. 60%, p = .87), as more patients with acute success of ablation presented with a relapse during follow-up (35.0 vs. 7.7%, p = .05). CONCLUSION: Acute success rate of FAT ablation might be improved by UHDM, without an adverse effect on procedural parameters. Nevertheless, further research is needed to understand the underlying mechanism for increased recurrence rates after acute successful ablation.
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Ablación por Catéter , Arritmias Cardíacas , Ablación por Catéter/efectos adversos , Ablación por Catéter/métodos , Humanos , Estudios Retrospectivos , Taquicardia , Resultado del TratamientoRESUMEN
Conduction disorders with need for permanent pacemaker (PPM) implantation remain frequent complications after transcatheter aortic valve implantation (TAVI). Up to 22% of PPM after TAVI are implanted for new onset left bundle branch block (LBBB) and atrioventricular block (AVB) I. However, clinical benefit and predictors of ventricular pacing in TAVI patients receiving PPM for this indication remain unclear. We retrospectively evaluated pacemaker interrogation data of patients who received a PPM post TAVI for new LBBB and new AVB I. The primary endpoint of this study was relevant ventricular pacing (ventricular pacing rate: Vp ≥ 1%) at the first outpatient pacemaker interrogation. Secondary endpoints were predictors for relevant ventricular pacing. At the first pacemaker interrogation (median follow up at 6.23 [2.8-14.8] months), median ventricular pacing frequency was 1.0% [0.1-17.8]. Out of 61 patients, 36 (59%) had Vp rates ≥ 1%. Patients with frequent ventricular pacing showed longer QRS duration (155 ms ± 17 ms vs. 144 ms ± 18 ms, p = 0.018) at the time of PPM implantation and were less likely treated with a balloon-expandable Edwards Sapiens Valve (39% vs. 12%, p = 0.040). Our findings suggest that the majority of patients with new LBBB and new AVB I after TAVI show relevant ventricular pacing rates at follow up. Further prospective studies are necessary to identify patients at higher risk of pacemaker dependency.
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Bloqueo Atrioventricular/complicaciones , Bloqueo Atrioventricular/terapia , Bloqueo de Rama/complicaciones , Bloqueo de Rama/terapia , Marcapaso Artificial , Reemplazo de la Válvula Aórtica Transcatéter/efectos adversos , Anciano , Anciano de 80 o más Años , Estimulación Cardíaca Artificial , Causalidad , Fenómenos Electrofisiológicos , Femenino , Prótesis Valvulares Cardíacas , Humanos , Masculino , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
OBJECTIVES: The heart rate score (HRS) serves as a device-based measure of impaired heart rate variability and is an independent predictor of death in patients with heart failure and a cardiac implantable electrical device. However, no data are available for predicting death from the HRS in patients with end stage heart failure and a left ventricular assist device. METHODS: From November 2011 to July 2018, a total of 56 patients with a pre-existing cardiac implantable electrical device underwent left ventricular assist device implantation at our 2 study sites. The ventricular HRS was calculated retrospectively during the first cardiac implantable electrical device follow-up examination following the index hospitalization. Survival during follow-up was correlated with initial HRS. RESULTS: During the follow-up period, 46.4% of the patients (n = 26) died. The median follow-up period was 33.2 months. The median HRS after the index hospitalization was 41.1 ± 21.8%. More patients with an HRS >65% died compared to patients with an HRS <30% (76.9% vs 14.4%; P = 0.007). CONCLUSIONS: In our multicentre experience, survival of patients after an left ventricular assist device implant correlates with the HRS. After confirmation of our findings in a larger cohort, the effect of rate-responsive pacing will be within the scope of further investigation.
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Insuficiencia Cardíaca , Trasplante de Corazón , Corazón Auxiliar , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Frecuencia Cardíaca , Humanos , Estudios Retrospectivos , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: It was recently demonstrated that the detection of atrial fibrillation based on heart rate tracking by optical sensors is feasible and reliable using the Apple Watch and the corresponding application. There are already a number of smartwatches and other wearable devices alongside the Apple Watch that can additionally record a single-lead electrocardiogram (ECG) and it is reasonable to expect this technology to become a standard feature, as is already the case with automated heart rate tracking. This could potentially have enormous impact regarding the early diagnosis of several cardiac diseases. CASE SUMMARY: A 61-year-old male patient without previously known coronary artery disease was admitted with subacute ST-elevation myocardial infarction (STEMI) caused by occlusion of left anterior descending artery. Due to mildness of symptoms, the patient did only seek medical attention due to morphological changes in the single-lead ECG tracing acquired on his Apple Watch 5. The ECG recording of his smartwatch clearly showed ST-elevation, QRS widening, R-wave loss, and T-wave inversion. Coronary angiography revealed occlusion of the left anterior descending and recanalization was performed. The patient recovered without any complications and was discharged from the hospital 4 days after admission. DISCUSSION: While the potential of ECG recordings by smartwatches to detect atrial fibrillation is currently under scientific investigation, this case highlights the possible potential of these devices to detect STEMI.
RESUMEN
Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides temporary cardiac and respiratory support and has emerged as an established salvage intervention for patients with hemodynamic compromise or shock. It is thereby used as a bridge to recovery, bridge to permanent ventricular assist devices, bridge to transplantation, or bridge to decision. However, weaning from VA-ECMO differs between centers, and information about standardized weaning protocols are rare. Given the high mortality of patients undergoing VA-ECMO treatment, it is all the more important to answer the many questions still remaining unresolved in this field Standardized algorithms are recommended to optimize the weaning process and determine whether the VA-ECMO can be safely removed. Successful weaning as a multifactorial process requires sufficient recovery of myocardial and end-organ function. The patient should be considered hemodynamically stable, although left ventricular function often remains impaired during and after weaning. Echocardiographic and invasive hemodynamic monitoring seem to be indispensable when evaluating biventricular recovery and in determining whether the VA-ECMO can be weaned successfully or not, whereas cardiac biomarkers may not be useful in stratifying those who will recover. This review summarizes the strategies of weaning of VA-ECMO and discusses predictors of successful and poor weaning outcome.
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The management of patients with a cardiac implantable electronic device (CIED) poses a particular challenge in the peri-interventional and perioperative medical environment due to the many forms of possible electromagnetic interference. Although the devices encountered nowadays are of increasing complexity, the vast majority of procedures can be safely performed in patients. The existing position statements and recommendations, however, have a low level of evidence and are often contradictory. In the context of intraoperative electrocauterization, one of the most important sources of electromagnetic interference in the medical environment, recent studies have suggested an increasingly pragmatically perioperative CIED management, which is not represented in the existing recommendations. This article gives an example of these newer findings and reports the currently used and appropriately adapted perioperative CIED management protocol. Extracorporeal shock wave lithotripsy was thought to cause severe interference in CIED patients based on older studies and in vitro experiments. Although electromagnetic interference is possible, clinical observations with modern devices show that the procedure can generally be safely applied in CIED patients. Physiotherapy often utilizes a variety of electromechanical devices, which can be a relevant source of electromagnetic interference. Although some of these therapies can be safely used, coordination with the responsible CIED unit is recommended.
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Desfibriladores Implantables , Fenómenos Electromagnéticos , Litotricia , Marcapaso Artificial , Humanos , Modalidades de FisioterapiaRESUMEN
BACKGROUND: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. METHODS: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. FINDINGS: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1ß as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1ß-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. INTERPRETATION: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. FUND: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research.
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Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Sepsis/metabolismo , Sepsis/fisiopatología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Activación Enzimática , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Especies Reactivas de Oxígeno/metabolismo , Sepsis/etiologíaRESUMEN
Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.
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Enfermedades Autoinmunes/inmunología , Movimiento Celular/inmunología , Trampas Extracelulares/inmunología , Midkina/inmunología , Miocarditis/inmunología , Miocardio/inmunología , Neutrófilos/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Movimiento Celular/genética , Trampas Extracelulares/genética , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/inmunología , Ratones , Ratones Transgénicos , Midkina/genética , Miocarditis/genética , Miocarditis/patología , Miocardio/patología , Neutrófilos/patología , Receptores de LDL/genética , Receptores de LDL/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Leukocyte-released antimicrobial peptides contribute to pathogen elimination and activation of the immune system. Their role in thrombosis is incompletely understood. Here we show that the cathelicidin LL-37 is abundant in thrombi from patients with acute myocardial infarction. Its mouse homologue, CRAMP, is present in mouse arterial thrombi following vascular injury, and derives mainly from circulating neutrophils. Absence of hematopoietic CRAMP in bone marrow chimeric mice reduces platelet recruitment and thrombus formation. Both LL-37 and CRAMP induce platelet activation in vitro by involving glycoprotein VI receptor with downstream signaling through protein tyrosine kinases Src/Syk and phospholipase C. In addition to acute thrombosis, LL-37/CRAMP-dependent platelet activation fosters platelet-neutrophil interactions in other inflammatory conditions by modulating the recruitment and extravasation of neutrophils into tissues. Absence of CRAMP abrogates acid-induced lung injury, a mouse pneumonia model that is dependent on platelet-neutrophil interactions. We suggest that LL-37/CRAMP represents an important mediator of platelet activation and thrombo-inflammation.
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Péptidos Catiónicos Antimicrobianos/química , Arterias/patología , Plaquetas/metabolismo , Inflamación/metabolismo , Neutrófilos/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/citología , Femenino , Humanos , Microscopía Intravital , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/química , Permeabilidad , Activación Plaquetaria , Transducción de Señal , CatelicidinasRESUMEN
Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.
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Arterias/patología , Células Endoteliales/inmunología , Hepacivirus/inmunología , Hepatitis C/tratamiento farmacológico , Células Mesangiales/inmunología , Proteína C/uso terapéutico , Enfermedades Vasculares/tratamiento farmacológico , Vénulas/patología , Animales , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/administración & dosificación , Trombosis , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismoRESUMEN
Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases.
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Plaquetas/metabolismo , Neoplasias Pulmonares/secundario , Pulmón/patología , Melanoma Experimental/patología , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Animales , Plaquetas/fisiología , Agregación Celular , Línea Celular Tumoral , Proliferación Celular , Pulmón/irrigación sanguínea , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Ratones , MicrocirculaciónRESUMEN
Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.
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ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Tromboplastina/biosíntesis , Animales , Coagulación Sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Trombosis/inducido químicamente , Trombosis/patología , Factor de von Willebrand/biosíntesisRESUMEN
INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.
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Plaquetas/metabolismo , Colágeno/metabolismo , FN-kappa B/metabolismo , Agregación Plaquetaria , Complejo de la Endopetidasa Proteasomal/metabolismo , Plaquetas/citología , Calcio/metabolismo , Humanos , Transducción de SeñalRESUMEN
In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.
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Citocinas/metabolismo , Células Endoteliales/virología , Hepacivirus/fisiología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Hepacivirus/genética , Interacciones Huésped-Patógeno , Humanos , Helicasa Inducida por Interferón IFIH1 , Rodamiento de Leucocito/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/farmacología , Interferencia de ARN , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
INTRODUCTION: Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. METHODS: Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. RESULTS: In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects. CONCLUSIONS: TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.