RESUMEN
OBJECTIVES: To develop a rodent model of persistent non-inflammatory bladder pain and to test macrophage migration inhibitory factor and high mobility box group 1 as mediators of bladder pain. METHODS: Female C57BL/6 mice received intravesical instillations of protease activated receptor 4 (100 µmol/L, for 1 h) three times every other day and abdominal mechanical hypersensitivity (50% mechanical threshold) was tested on day 0 (baseline), and at days 1, 2, 3, 4, 7 and 9 after the first protease-activated receptor 4 injection. At the end of the experiment, micturition changes were measured and bladders were examined for histological changes. Macrophage migration inhibitory factor antagonist (MIF098; 40 mg/kg i.p. b.i.d.) or high mobility group box 1 inhibitor (glycyrrhizin; 50 mg/kg i.p. daily) was administered from day 2 until day 8. RESULTS: There was a significant and persistent decrease in abdominal mechanical threshold starting from day 3 in the protease-activated receptor 4-treated group that persisted until day 9 (5 days post-last instillation), but not in the control group. Glycyrrhizin fully reversed while MIF098 partially reversed abdominal mechanical hypersensitivity in protease-activated receptor 4-treated mice. The changes started on day 3 after the first protease-activated receptor 4 instillation, and analgesic effects lasted throughout the rest of the testing period. None of the groups had significant micturition changes or overt bladder histological changes. CONCLUSIONS: Repeated intravesical protease activated receptor 4 instillations produce persistent bladder pain without inflammation. Macrophage migration inhibitory factor and high mobility group box 1 are possible effective target molecules for bladder pain alleviation.
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Modelos Animales de Enfermedad , Proteína HMGB1/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Dolor Pélvico/patología , Receptores de Trombina/administración & dosificación , Administración Intravesical , Animales , Femenino , Ácido Glicirrínico/farmacología , Ácido Glicirrínico/uso terapéutico , Proteína HMGB1/antagonistas & inhibidores , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Dolor Pélvico/inducido químicamente , Dolor Pélvico/tratamiento farmacológico , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Type 2 myocardial infarction (MI) is characterized by an imbalance between myocardial blood supply and demand, leading to myocardial ischemia without coronary plaque rupture, but its diagnosis is challenging. METHODS: In the TRACER trial, patients with non-ST-segment elevation acute coronary syndromes were included. We aimed to describe provoking factors, cardiac biomarker profiles, treatment patterns, and clinical outcomes of patients with type 2 MIs. MI events during trial follow-up were adjudicated by an independent clinical events classification committee (CEC) and were classified according to the Third Universal Definition of MI. Using available source documents retrieved as part of the CEC process, we performed a retrospective chart abstraction to collect details on the type 2 MIs. Cox regression models were used to explore the association between MI type (type 1 or type 2) and cardiovascular death. RESULTS: Overall, 10.3% (n=1327) of TRACER participants had a total of 1579 adjudicated MIs during a median follow-up of 502 days (25th and 75th percentiles [IQR] 349-667). Of all MIs, 5.2% (n=82) were CEC-adjudicated type 2 MIs, occurring in 76 patients. The incidence of type 2 MI was higher in the first month following randomization, after which the distribution became more scattered. The most frequent potential provoking factors for type 2 MIs were tachyarrhythmias (38.2%), anemia/bleeding (21.1%), hypotension/shock (14.5%), and hypertensive emergencies (11.8%). Overall, 36.3% had a troponin increase >10× the upper limit of normal. Coronary angiography was performed in 22.4% (n=17) of patients during hospitalizations due to type 2 MIs. The hazard of cardiovascular death was numerically higher following type 2 MI (vs. no MI, adj. HR 11.82, 95% CI 5.71-24.46; P<.0001) than that of type 1 MI (vs. no MI, adj. HR 8.90, 95% CI 6.93-11.43; P<.0001). CONCLUSIONS: Type 2 MIs were more prevalent in the first month after ACS, were characterized by the presence of triggers and infrequent use of an invasive strategy, and were associated with a high risk of death. Further efforts are needed to better define the role and implications of type 2 MI in both clinical practice and research.
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Síndrome Coronario Agudo/tratamiento farmacológico , Síndrome Coronario Agudo/mortalidad , Infarto del Miocardio sin Elevación del ST/tratamiento farmacológico , Infarto del Miocardio sin Elevación del ST/mortalidad , Receptores de Trombina/antagonistas & inhibidores , Síndrome Coronario Agudo/diagnóstico por imagen , Anciano , Angiografía Coronaria/métodos , Progresión de la Enfermedad , Método Doble Ciego , Electrocardiografía/métodos , Femenino , Estudios de Seguimiento , Humanos , Internacionalidad , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/diagnóstico por imagen , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/mortalidad , Infarto del Miocardio sin Elevación del ST/diagnóstico por imagen , Receptores de Trombina/administración & dosificación , Medición de Riesgo , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The availability of direct oral anticoagulants has caused a paradigm shift in thrombosis management. The direct thrombin inhibitor dabigatran seems to obstruct tenase complex by inhibiting thrombin generated in the initial phase and feed back to the amplification phase of cell-based coagulation reactions. However, it is still not fully understood if and how dabigatran impact platelet function. This observational study aimed to assess in vitro platelet function in patients with atrial fibrillation receiving dabigatran. Platelet aggregability was tested with platelet-rich plasma using platelet aggregometry (PACKS-4 aggregometer). Blood samples were stimulated with thrombin receptor agonist peptide (TRAP; 32 µmol/L). RESULTS: A total of 28 patients with nonvalvular atrial fibrillation were enrolled. The mean age was 71.57 (9.75) years (range: 50-87 years), 16 patients were women, and the mean CHA2DS2VASc score was 3.93 (1.41). All patients began treatment with dabigatran as initial anticoagulant treatment. The minimum term use of dabigatran was 18 days. Dabigatran doses were 110 mg (57.14%) or 150 mg (42.86%) twice daily. The TRAP-induced platelet aggregation was significantly lower 2 hours after taking dabigatran compared to baseline value (79.39 [13.38] vs 90.14 [10.5]). CONCLUSION: The TRAP-induced platelet aggregation was reduced in cardiovascular patients 2 hours after receiving dabigatran. Our findings could have some important clinical implications because platelet aggregation and coagulation cascade are affected at the same time.
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Dabigatrán/administración & dosificación , Interacciones Farmacológicas , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/administración & dosificación , Anciano , Anciano de 80 o más Años , Anticoagulantes , Antitrombinas , Dabigatrán/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
PURPOSE: The aim of the study was to evaluate the potential influence of ranibizumab and bevacizumab on platelet activation and aggregation, which are critical processes in the pathogenesis of arterial thromboembolic events (ATEs). METHODS: For the assessment of platelet function, flow cytometry and aggregometry were employed. Platelets were isolated from healthy volunteers and exposed to ranibizumab (1 mg/ml and 150 ng/ml) and bevacizumab (2.5 mg/ml and 3 µg/ml) or their solvents for 10 and 30 min prior to the addition of TRAP (25 µM), PAR-4-AP (25 µM) or thrombin (0.02 U/ml). The surface expression of activated GP IIb/IIIa, P-selectin (CD62P) and platelet-bound stromal cell-derived factor-1 (SDF-1) was measured on resting (nonactivated) and activated platelets by flow cytometry. The platelet aggregation capacity was examined using light transmission aggregometry. RESULTS: The expression of surface activation markers did not differ significantly between nonstimulated and TRAP-, PAR-4-AP- or thrombin-activated platelets after incubating with ranibizumab. However, GP IIb/IIIa, CD62P and SDF-1 were significantly downregulated in PAR-4-AP- and thrombin-activated platelets after exposure to bevacizumab 2.5 mg/ml. In addition, ranibizumab- and bevacizumab-FITC were significantly increased in all activated platelets. No significant differences were observed in the aggregation of activated platelets after incubation with ranibizumab or bevacizumab. CONCLUSION: All ranibizumab concentrations as well as the bevacizumab concentration of 3 µg/ml had no influence on platelet activation and aggregation. Therefore, this in vitro study did not show any relationship between the exposition of activated platelets to ranibizumab or bevacizumab and the development of ATEs. However, the highest level of bevacizumab interfered with platelet activation, leading to downregulation of platelet activation markers. This observation might explain why the systemic treatment with high-dose bevacizumab could be associated with an increased risk of bleeding. Regarding the use of lower intravitreal dosages, further research should focus on the complex interactions between platelets and other cells, such as endothelial cells, which might stronger relate to a potentially increased risk of ATEs and depend on systemic vascular endothelial growth factor levels. Facing the different activation profiles, the diverse effects of the drugs on the cellular level have to be critically scrutinized for their clinical relevance.
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Inhibidores de la Angiogénesis/farmacología , Bevacizumab/farmacología , Plaquetas/efectos de los fármacos , Activación Plaquetaria/fisiología , Ranibizumab/farmacología , Plaquetas/metabolismo , Quimiocina CXCL12/metabolismo , Citometría de Flujo , Humanos , Selectina-P/metabolismo , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Trombina/administración & dosificación , Trombina/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.
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Plaquetas/metabolismo , Factor V/biosíntesis , Regulación de la Expresión Génica , Receptor PAR-1/fisiología , Receptores de Trombina/fisiología , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Factor V/genética , Regulación de la Expresión Génica/fisiología , Humanos , Receptor PAR-1/administración & dosificación , Receptores de Trombina/administración & dosificación , Trombina/antagonistas & inhibidores , Trombina/biosíntesisRESUMEN
BACKGROUND AND PURPOSE: P2Y(1) is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy. EXPERIMENTAL APPROACH: Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day(-1) ) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y(1 ) inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed. KEY RESULTS: Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α(IIb) ß(3) activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α(IIb) b(3) activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s(-1) ), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y(12) inhibitor 2-MeSAMP. CONCLUSIONS AND IMPLICATIONS: Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y(1) in the blood of patients with CAD appears to play a more important role than previously anticipated.
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Aspirina/farmacología , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2Y/farmacología , Ticlopidina/análogos & derivados , Adenosina Difosfato/administración & dosificación , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Anciano , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Clopidogrel , Colágeno/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Purinérgicos P2Y1/efectos de los fármacos , Receptores de Trombina/administración & dosificación , Trombosis/prevención & control , Ticlopidina/farmacologíaRESUMEN
Solulin is a novel recombinant soluble derivative of human thrombomodulin. In this first human study of Solulin, the safety, tolerability, pharmacokinetics and pharmacodynamics of Solulin in 30 healthy volunteers in response to single (0.6-30 mg) and 12 healthy volunteers in response to multiple (1 and 10 mg) ascending intravenous bolus doses compared to placebo are described. Solulin was shown to be well tolerated, and demonstrated linear pharmacokinetics over the clinically relevant dose range, with a plasma elimination half-life of 15-30 hours, indicating that a less than daily dose may be required for therapeutic use. Steady-state plasma levels after multiple dosing were reached after 48 hours. Solulin has shown to be able to inhibit thrombin generation without increasing levels of aPC/PCI complexes. Coagulation parameters INR and PT were not changed, aPTT was elevated to about 10% above the upper limit of normal after the highest single dose only. Thrombin clotting time was prolonged after administration of high dose Solulin (10, 30 mg). No effect on in vitro bleeding time has been found. There was no evidence of bleeding risk with Solulin administration. The pharmacodynamic effects correlated with Solulin plasma concentrations. This demonstrates that the antithrombotic effect of Solulin is predictable, suggesting that patient monitoring is not expected. The results of this study provide evidence that Solulin can be expected to be an effective and safe anticoagulant, and further clinical investigation is warranted.
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Anticoagulantes/farmacocinética , Coagulación Sanguínea/efectos de los fármacos , Fibrinolíticos/farmacocinética , Proteínas Recombinantes/farmacocinética , Adolescente , Adulto , Anticoagulantes/administración & dosificación , Anticoagulantes/efectos adversos , Disponibilidad Biológica , Pruebas de Coagulación Sanguínea , Relación Dosis-Respuesta a Droga , Fibrinolíticos/administración & dosificación , Fibrinolíticos/efectos adversos , Semivida , Hemorragia/inducido químicamente , Humanos , Inyecciones Intravenosas , Relación Normalizada Internacional , Masculino , Tasa de Depuración Metabólica , Países Bajos , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Receptores de Trombina/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Medición de Riesgo , Adulto JovenRESUMEN
Active thrombin is found in the airways of patients with a variety of inflammatory lung diseases. However, whether thrombin contributes to the pathologies of these diseases is unknown, although thrombin is a potent inflammatory mediator in other organ systems. In the present study we have assessed the acute inflammatory effect of inhaled thrombin and investigated the possible receptors mediating any effects in mice. Thrombin (200-2000 U kg(-1) intranasally), induced the recruitment of a small, but significant, number of neutrophils into the airways as assessed by differential counts of cells retrieved by bronchoalveolar lavage (BAL). This small response was mimicked by peptide agonists of proteinase-activated receptor-4 (PAR(4); GYPGKF, AYPGKF; 2-20 mg kg(-1)), but not PAR(1) (SFLLRN; 2-20 mg kg(-1)). By contrast, trypsin (200-2000 U kg(-1)) caused profound inflammation and lung damage. Concentrations of tumour necrosis factor-alpha (TNF-alpha) were elevated in BAL fluid from thrombin-treated mice, and a TNF-alpha-neutralising antibody inhibited the influx of neutrophils in response to thrombin. Although isolated alveolar macrophages appeared to express PAR(1)- and PAR(4)-immunoreactivity, these cells failed to release TNF-alpha above baseline levels in response to thrombin, trypsin or any of the peptide PAR agonists. Neither thrombin (2000 U kg(-1)) nor trypsin (200 U kg(-1)) modified the airway neutrophilia in response to intranasal bacterial lipopolysaccharide (LPS; 100 micrograms kg(-1)). In conclusion, exogenous thrombin has only a modest acute inflammatory action in the lung that appears to be mediated by PAR(4) and involve release of TNF-alpha from an unknown source.
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Receptores de Trombina/administración & dosificación , Receptores de Trombina/agonistas , Administración por Inhalación , Administración Intranasal , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Oligopéptidos/administración & dosificación , Oligopéptidos/agonistas , Oligopéptidos/farmacocinética , Fragmentos de Péptidos/administración & dosificación , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/ultraestructura , Receptor PAR-1/análisis , Receptor PAR-1/efectos de los fármacos , Receptor PAR-2/análisis , Receptor PAR-2/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Trombina/administración & dosificación , Trombina/antagonistas & inhibidores , Trombina/farmacocinética , Tráquea/patología , Tripsina/administración & dosificación , Tripsina/efectos adversos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Reino UnidoRESUMEN
Activation of colonic proteinase-activated receptor-2 (PAR-2) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is under debate and could be neurogenic and/or the consequence of tight-junction opening with passage of exogenous pathogens into the lamina propria. The present study aimed to further characterize the inflammatory effect of PAR-2 activation by investigating: 1) the role of NO, 2) the role of afferent neurons, and 3) a possible cause and effect relationship between colonic paracellular permeability changes and mucosal inflammation. Thus, intracolonic infusion to mice of the PAR-2-activating peptide, SLIGRL, increased both myeloperoxidase (MPO) activity and damage scores indicating colonic inflammation, and enhanced colonic permeability to (51)Cr-EDTA from 2 to 4 h after its infusion. NO synthase inhibitors, L-NAME and aminoguanidine, as well as the neurotoxin capsaicin and NK1, calcitonin gene-related peptide (CGRP) receptor antagonists, SR140333 and CGRP(8-37), prevented SLIGRL-induced MPO and damage score increases and permeability. In contrast, although the tight-junction blocker, 2,4,6-triaminopyrimidine, and the myosin L chain kinase inhibitor, ML-7, prevented SLIGRL-induced increase in permeability, they did not prevent MPO and damage score increases. Taken together our data show that both NO and capsaicin-sensitive afferent neurons are involved in PAR-2-mediated colonic inflammation and paracellular permeability increase. Nevertheless, the inflammation process is not a consequence of increased permeability which results at least in part from the activation of myosin L chain kinase.
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Permeabilidad de la Membrana Celular , Colon/patología , Mucosa Intestinal/metabolismo , Neuronas Aferentes/fisiología , Óxido Nítrico/fisiología , Receptores de Trombina/administración & dosificación , Receptores de Trombina/fisiología , Administración Rectal , Animales , Antígenos , Antígenos de Superficie , Azepinas/administración & dosificación , Azepinas/farmacología , Péptido Relacionado con Gen de Calcitonina/farmacología , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/enzimología , Colon/metabolismo , Desnervación , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Inflamación/enzimología , Inflamación/metabolismo , Inflamación/fisiopatología , Inyecciones Intraperitoneales , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Lectinas Tipo C , Masculino , Ratones , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Subfamilia B de Receptores Similares a Lectina de Células NK , Naftalenos/administración & dosificación , Naftalenos/farmacología , Neuronas Aferentes/patología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Oligopéptidos/administración & dosificación , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Proteínas/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Quinuclidinas/administración & dosificación , Quinuclidinas/farmacología , Receptor PAR-2RESUMEN
Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1 beta, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-D-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.
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Complejo SIDA Demencia/metabolismo , Astrocitos/metabolismo , Receptores de Trombina/biosíntesis , Regulación hacia Arriba , Complejo SIDA Demencia/enzimología , Complejo SIDA Demencia/fisiopatología , Secuencia de Aminoácidos , Animales , Astrocitos/enzimología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Encéfalo/metabolismo , Sistema Libre de Células/fisiología , Cuerpo Estriado/inmunología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiología , Implantes de Medicamentos , Feto , VIH-1/fisiología , Humanos , Interleucina-1/biosíntesis , Masculino , Ratones , Datos de Secuencia Molecular , Esclerosis Múltiple/metabolismo , Neuronas/metabolismo , Neuronas/patología , Neurotoxinas/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Péptidos/administración & dosificación , Péptidos/fisiología , Receptor PAR-1 , Receptores de N-Metil-D-Aspartato/fisiología , Receptores de Trombina/administración & dosificación , Receptores de Trombina/agonistas , Receptores de Trombina/fisiología , Trombina/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The proteinase-activated receptor 2 is expressed on a subset of primary afferent neurons and may participate in the neurogenic component of inflammation. We hypothesized that this receptor may also play a role in neuronal sensitization and contribute to the pathogenesis of pain in inflammatory conditions such as pancreatitis. Using a specific proteinase-activated receptor 2 activating peptide, we found evidence of such sensitization in vitro in the form of enhanced capsaicin- and KCl-evoked release of calcitonin gene-related peptide, a marker for nociceptive signaling. We then demonstrated that injection of the proteinase-activated receptor 2 activating peptide into the pancreatic duct can activate and sensitize pancreas-specific afferent neurons in vivo, as measured by Fos expression in the dorsal horn of the spinal cord. These observations suggest that proteinase-activated receptor 2 contributes to nociceptive signaling and may provide a novel link between inflammation and pain.
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Ganglios Espinales/metabolismo , Neuronas Aferentes/metabolismo , Dolor/fisiopatología , Receptores de Trombina/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina , Células Cultivadas , Vías de Administración de Medicamentos , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Masculino , Neuronas Aferentes/efectos de los fármacos , Dolor/inducido químicamente , Dimensión del Dolor , Páncreas/citología , Páncreas/efectos de los fármacos , Páncreas/inervación , Conductos Pancreáticos/efectos de los fármacos , Células del Asta Posterior/metabolismo , Cloruro de Potasio , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor PAR-2 , Receptores de Trombina/administración & dosificación , Receptores de Trombina/agonistasRESUMEN
Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 +/- 11 to 71 +/- 24% (p < 0.05) and decreased mean rolling velocity from 63 +/- 29 to 32 +/- 2 micrometer/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 +/- 8 to 30 +/- 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0-15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 +/- 3 vs 30 +/- 6%, p < 0.05) and mean rolling velocity was higher (67 +/- 46 vs 52 +/- 36 micrometer/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.
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Peritonitis/enzimología , Peritonitis/genética , Receptores de Trombina/deficiencia , Receptores de Trombina/genética , Animales , Adhesión Celular/genética , Adhesión Celular/inmunología , Línea Celular , Movimiento Celular/genética , Movimiento Celular/inmunología , Endotelio Vascular , Femenino , Hemodinámica/genética , Hemodinámica/inmunología , Humanos , Leucocitos/inmunología , Leucocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía por Video , Músculo Esquelético/irrigación sanguínea , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Activación Neutrófila/genética , Activación Neutrófila/inmunología , Oligopéptidos/administración & dosificación , Oligopéptidos/inmunología , Peritonitis/inmunología , Peritonitis/fisiopatología , Receptor PAR-2 , Receptores de Trombina/administración & dosificación , Receptores de Trombina/agonistasRESUMEN
Proteinase-activated receptor 2 (PAR2) has been suggested to play a role in inflammatory reactions. Because leukocyte-endothelial cell interactions are critical events during inflammatory reactions, and because PAR2 is expressed both on endothelium and leukocytes, we have examined the effects of PAR2-activating peptides (PAR2-APs) on leukocyte rolling and adhesion in mesenteric venules and on leukocyte recruitment into the peritoneal cavity. Using intravital microscopy, leukocyte rolling, flux, and adhesion in rat mesenteric postcapillary venules were quantified. Topical addition of PAR2-APs (10 microM) for 1 min to the superfused venule induced a significant increase in leukocyte rolling and adherence. The increase in leukocyte adherence was not affected by pretreatment with a mast cell stabilizer (sodium cromoglycate) nor by prior degranulation of mast cells with compound 48/80. Nonetheless, both leukocyte rolling and adhesion were completely inhibited by pretreatment with a platelet-activating factor receptor antagonist (WEB 2086). Intraperitoneal injections of a selective PAR2-AP (SLIGRL-NH2) caused a significant increase in leukocyte migration into the peritoneal cavity. The effect of SLIGRL-NH2 on peritoneal leukocyte infiltration was completely inhibited by WEB 2086. These data suggest that PAR2 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence, and recruitment, by a mechanism dependent on platelet-activating factor release.
Asunto(s)
Movimiento Celular/fisiología , Endopeptidasas/metabolismo , Leucocitos/fisiología , Péptidos/administración & dosificación , Receptores de Trombina/administración & dosificación , Animales , Adhesión Celular/fisiología , Inyecciones Intraperitoneales , Masculino , Mastocitos/fisiología , Péptidos/fisiología , Factor de Activación Plaquetaria/fisiología , Ratas , Ratas Wistar , Receptor PAR-2 , Receptores de Trombina/fisiologíaRESUMEN
BACKGROUND: The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS: Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.