RESUMEN
Activated factor X is a key component of the coagulation cascade, but whether it directly regulates pathological cardiac remodeling is unclear. In mice subjected to pressure overload stress, cardiac factor X mRNA expression and activity increased concurrently with cardiac hypertrophy, fibrosis, inflammation and diastolic dysfunction, and responses blocked with a low coagulation-independent dose of rivaroxaban. In vitro, neurohormone stressors increased activated factor X expression in both cardiac myocytes and fibroblasts, resulting in activated factor X-mediated activation of protease-activated receptors and pro-hypertrophic and -fibrotic responses, respectively. Thus, inhibition of cardiac-expressed activated factor X could provide an effective therapy for the prevention of adverse cardiac remodeling in hypertensive patients.
RESUMEN
Type 2 Diabetes Mellitus (T2DM) is the most prevalent form of diabetes in the USA, thus, the identification of biomarkers that could be used to predict the progression from prediabetes to T2DM would be greatly beneficial. Recently, circulating RNA including microRNAs (miRNAs) present in various body fluids have emerged as potential biomarkers for various health conditions, including T2DM. Whereas studies that examine the changes of miRNA spectra between healthy controls and T2DM individuals have been reported, the goal of this study is to conduct a baseline comparison of prediabetic individuals who either progress to T2DM, or remain prediabetic. Using an advanced small RNA sequencing library construction method that improves the detection of miRNA species, we identified 57 miRNAs that showed significant concentration differences between progressors (progress from prediabetes to T2DM) and non-progressors. Among them, 26 have been previously reported to be associated with T2DM in either body fluids or tissue samples. Some of the miRNAs identified were also affected by obesity. Furthermore, we identified miRNA panels that are able to discriminate progressors from non-progressors. These results suggest that upon further validation these miRNAs may be useful to predict the risk of conversion to T2DM from prediabetes.
Asunto(s)
Biomarcadores/sangre , Ácidos Nucleicos Libres de Células/sangre , Diabetes Mellitus Tipo 2/diagnóstico , MicroARNs/sangre , Anciano , Estudios de Casos y Controles , Ácidos Nucleicos Libres de Células/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , PronósticoRESUMEN
INTRODUCTION: Rivaroxaban selectively inhibits factor Xa (FXa), which plays a central role in blood coagulation. In addition, FXa activates protease-activated receptor-2 (PAR-2). We have shown that PAR-2-/- mice exhibit less cardiac dysfunction after cardiac injury. MATERIAL AND METHODS: Wild-type (WT) and PAR-2-/- mice were subjected to left anterior descending artery (LAD) ligation to induce cardiac injury and heart failure. Mice received either placebo or rivaroxaban chow either starting at the time of surgery or 3â¯days after surgery and continued up to 28â¯days. Cardiac function was measured by echocardiography pre-surgery and 3, 7 and 28â¯days after LAD ligation. We also measured anticoagulation, intravascular thrombi, infarct size, cardiac hypertrophy and inflammation at various times. RESULTS: Rivaroxaban increased the prothrombin time and inhibited the formation of intravascular thrombi in mice subjected to LAD ligation. WT mice receiving rivaroxaban immediately after surgery had similar infarct sizes at day 1 as controls but exhibited significantly less impairment of cardiac function at day 3 and beyond compared to the placebo group. Rivaroxaban also inhibited the expansion of the infarct at day 28. Rivaroxaban did not significantly affect the expression of inflammatory mediators or a neutrophil marker at day 2 after LAD ligation. Delaying the start of rivaroxaban administration until 3â¯days after surgery failed to preserve cardiac function. In addition, rivaroxaban did not reduce cardiac dysfunction in PAR-2-/- mice. CONCLUSIONS: Early administration of rivaroxaban preserves cardiac function in mice after LAD ligation.
Asunto(s)
Inhibidores del Factor Xa/uso terapéutico , Cardiopatías/tratamiento farmacológico , Infarto del Miocardio/prevención & control , Rivaroxabán/uso terapéutico , Animales , Modelos Animales de Enfermedad , Inhibidores del Factor Xa/farmacología , Humanos , Ratones , Rivaroxabán/farmacologíaRESUMEN
A decline in ß-cell function is a prerequisite for the development of type 2 diabetes, yet the level of ß-cell function in individuals at risk of the condition is rarely measured. This is due, in part, to the fact that current methods for assessing ß-cell function are inaccurate, prone to error, labor-intensive, or affected by glucose-lowering therapy. The aim of the current study was to identify novel circulating biomarkers to monitor ß-cell function and to identify individuals at high risk of developing ß-cell dysfunction. In a nested case-control study from the Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) cohort (n = 1157), proteomics and miRNA profiling were performed on fasting plasma samples from 43 individuals who progressed to impaired glucose tolerance (IGT) and 43 controls who maintained normal glucose tolerance (NGT) over three years. Groups were matched at baseline for age, gender, body mass index (BMI), insulin sensitivity (euglycemic clamp) and ß-cell glucose sensitivity (mathematical modeling). Proteomic profiling was performed using the SomaLogic platform (Colorado, USA); miRNA expression was performed using a modified RT-PCR protocol (Regulus Therapeutics, California, USA). Results showed differentially expressed proteins and miRNAs including some with known links to type 2 diabetes, such as adiponectin, but also novel biomarkers and pathways. In cross sectional analysis at year 3, the top differentially expressed biomarkers in people with IGT/ reduced ß-cell glucose sensitivity were adiponectin, alpha1-antitrypsin (known to regulate adiponectin levels), endocan, miR-181a, miR-342, and miR-323. At baseline, adiponectin, cathepsin D and NCAM.L1 (proteins expressed by pancreatic ß-cells) were significantly lower in those that progressed to IGT. Many of the novel prognostic biomarker candidates were within the epithelial-mesenchymal transition (EMT) pathway: for example, Noggin, DLL4 and miR-181a. Further validation studies are required in additional clinical cohorts and in patients with type 2 diabetes, but these results identify novel pathways and biomarkers that may have utility in monitoring ß-cell function and/ or predicting future decline, allowing more targeted efforts to prevent and intercept type 2 diabetes.
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Glucemia/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Intolerancia a la Glucosa/sangre , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Diabetes Mellitus Tipo 2/sangre , Diagnóstico Precoz , Femenino , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Proteases and proteinase-activated receptor (PAR) activation are involved in several intestinal inflammatory conditions. We hypothesized that serine proteases and PAR activation could also modulate the intestinal injury induced by ischemia-reperfusion (I-R). C57Bl/6 mice were subjected to 90 minutes of intestinal ischemia followed or not by reperfusion. Sham-operated animals served as controls. After ischemia, plasma and tissue serine protease activity levels were increased compared to the activity measured in plasma and tissues from sham-operated mice. This increase was maintained or further enhanced after 2 and 5 hours of reperfusion, respectively. Trypsin (25 kDa) was detected in tissues both after ischemia and 2 hours of reperfusion. Treatment with FUT-175 (10 mg/kg), a potent serine protease inhibitor, increased survival after I-R, inhibited tissue protease activity, and significantly decreased intestinal myeloperoxidase (MPO) activity and chemokine and adhesion molecule expression. We investigated whether serine proteases modulate granulocyte recruitment by a PAR-dependent mechanism. MPO levels and adhesion molecule expression were significantly reduced in I-R groups pre-treated with the PAR(1) antagonist SCH-79797 (5 mg/kg) and in Par(2)(-/-)mice, compared, respectively, to vehicle-treated group and wild-type littermates. Thus, increased proteolytic activity and PAR activation play a pathogenic role in intestinal I-R injury. Inhibition of PAR-activating serine proteases could be beneficial to reduce post-ischemic intestinal inflammation.
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Granulocitos/fisiología , Intestino Delgado/irrigación sanguínea , Isquemia/enzimología , Daño por Reperfusión/prevención & control , Inhibidores de Serina Proteinasa/farmacología , Animales , Benzamidinas , Quimiocinas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Granulocitos/enzimología , Guanidinas/farmacología , Isquemia/patología , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Inhibidores de Proteasas/farmacología , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/patología , Tripsina/metabolismo , alfa-Macroglobulinas/metabolismoRESUMEN
Rivaroxaban, an oral, direct factor Xa inhibitor, has been approved in several countries for thromboprophylaxis after elective hip or knee arthroplasty based on favorable benefit-risk profile and improved efficacy compared to enoxaparin in reducing the composite of symptomatic and asymptomatic deep vein thrombosis, nonfatal pulmonary embolism, and all-cause mortality. Given the potential therapeutic utility of factor Xa inhibition in arterial thrombosis, we evaluated the antithrombotic activity of rivaroxaban in a model of arterial thrombosis in anesthetized rats in which thrombotic occlusion was induced by electrolytic injury of the carotid artery. Rivaroxaban, 0.3, 1 or 3âmg/kg, enoxaparin, 10âmg/kg, or vehicle were infused intravenously to anesthetized rats and time to occlusion as well as coagulation parameters monitored following carotid electrolytic injury. Although the lowest dose of rivaroxaban (0.3âmg/kg) did not prolong occlusion time compared to vehicle, rivaroxaban at 1 or 3âmg/kg prevented occlusion in all vessels during the 30-min observation period (median occlusion time >30âmin), which was greater than that following a single dose of enoxaparin infused at a dose of 10âmg/kg (median time to occlusion = 21.6âmin). Rivaroxaban was also effective following oral dosing at 3âmg/kg. Rivaroxaban's antithrombotic activity was paralleled by dose-dependent increases in prothrombin time (PT) and activated clotting time (ACT) without significant changes in activated partial thromboplastin time. Rivaroxaban also markedly increased Russell's viper venom time (RVVT) and decreased thrombin-antithrombin complex concentrations at all doses. These findings support the potential utility of rivaroxaban in arterial thrombotic disorders such as acute coronary syndrome, stroke and peripheral arterial disease.
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Coagulación Sanguínea/efectos de los fármacos , Traumatismos de las Arterias Carótidas/sangre , Oclusión Coronaria/sangre , Trombosis Coronaria/sangre , Inhibidores del Factor Xa , Morfolinas/administración & dosificación , Tiofenos/administración & dosificación , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/uso terapéutico , Traumatismos de las Arterias Carótidas/inducido químicamente , Oclusión Coronaria/inducido químicamente , Trombosis Coronaria/inducido químicamente , Modelos Animales de Enfermedad , Electrólitos/efectos adversos , Enoxaparina/administración & dosificación , Enoxaparina/uso terapéutico , Factor Xa/metabolismo , Fibrinolíticos/administración & dosificación , Fibrinolíticos/uso terapéutico , Inyecciones Intravenosas , Masculino , Morfolinas/uso terapéutico , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Ratas , Ratas Sprague-Dawley , Rivaroxabán , Tiofenos/uso terapéuticoRESUMEN
OBJECTIVE: Our aim was to determine the contribution of proteinase-activated receptor-2 (PAR(2))-expressing bone marrow-derived cells on the development of colonic inflammation. MATERIALS: Chimeric mice were generated by injecting bone marrow cells from wildtype (PAR (2) (+/+) ) or PAR(2) knockout mice (PAR (2) (-/-) ) into irradiated PAR (2) (+/+) or PAR (2) (-/-) mice. TREATMENTS: Colitis was induced by giving 2.5% dextran sodium sulfate (DSS) solution for 7 days or by a single intracolonic administration of trinitrobenzene sulphonic acid (TNBS, 2 mg dissolved in 40% ethanol). METHODS: Seven days after the induction of colitis, bowel thickness, inflammatory parameters [myeloperoxidase (MPO) activity, macroscopic/microscopic damage scores], and leukocyte trafficking (visualized via intravital microscopy) were assessed. RESULTS: Total deficiency of PAR(2) resulted in a marked reduction in severity of both TNBS and DSS induced colitis as assessed by MPO activity, macroscopic damage, bowel thickness, and leukocyte adherence. Colitis was attenuated in all chimeric lines in which there was loss of PAR(2) in the host, non-bone marrow-derived tissue, independent of the status of PAR expression by bone marrow-derived cells. Interestingly, TNBS colitis was attenuated in PAR (2) (+/+) chimeric mice with PAR (2) (-/-) derived bone marrow but these animals were not protected from DSS colitis. CONCLUSIONS: Expression of PAR(2) by host-derived tissues plays a dominant role in regulating colonic inflammation. PAR(2) expression by bone marrow-derived cells appears to play a role in TNBS colitis but not in DSS induced injury.
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Colitis/metabolismo , Receptor PAR-2/metabolismo , Animales , Células de la Médula Ósea/efectos de la radiación , Trasplante de Médula Ósea , Quimera , Colitis/inducido químicamente , Colitis/patología , Colon/patología , Colon/efectos de la radiación , Sulfato de Dextran/toxicidad , Inflamación , Leucocitos/patología , Leucocitos/efectos de la radiación , Ratones , Ratones Noqueados , Peroxidasa/análisis , Receptor PAR-2/análisis , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.
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Anticoagulantes/síntesis química , Fibrinolíticos/síntesis química , Guanidinas/síntesis química , Pirazinas/síntesis química , Trombina/antagonistas & inhibidores , Secuencias de Aminoácidos , Animales , Anticoagulantes/farmacocinética , Anticoagulantes/farmacología , Presión Sanguínea/efectos de los fármacos , Células CACO-2 , Cristalografía por Rayos X , Citocromo P-450 CYP3A , Inhibidores del Citocromo P-450 CYP3A , Perros , Método Doble Ciego , Electrocardiografía , Femenino , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacología , Guanidinas/farmacocinética , Guanidinas/farmacología , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Pirazinas/farmacocinética , Pirazinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-Actividad , Trombina/química , Trombosis de la Vena/sangre , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
Whereas heparin functions as an antithrombotic agent by promoting antithrombin III-based inhibition of thrombin and factor Xa, there is less appreciation for the combination behavior with small-molecule, direct inhibitors of these proteases. We conducted a study in a high-shear arterial environment to explore the potential for a cooperative antithrombotic effect with a thrombin inhibitor (argatroban), a factor Xa inhibitor (YM-60828), and a dual thrombin/factor Xa inhibitor (RWJ-445167). We employed a platelet-dependent vascular injury model in which rats were subjected to an acute electrical injury to the carotid artery. Antithrombotic efficacy was measured for thrombin inhibitor argatroban and factor Xa inhibitor YM-60828 administered alone or in combination. The results indicate that there is a cooperative antithrombotic effect in vivo when both thrombin and factor Xa are inhibited simultaneously. The dual thrombin/factor Xa inhibitor RWJ-445167 was found to have potent antithrombotic activity in this high-shear environment. A comparison of results for RWJ-445167 and argatroban showed additional efficacy with RWJ-445167, suggestive of drug synergy.
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Inhibidores del Factor Xa , Guanidinas/farmacología , Naftalenos/farmacología , Ácidos Pipecólicos/farmacología , Piperidinas/farmacología , Sulfonamidas/farmacología , Trombina/antagonistas & inhibidores , Animales , Arginina/análogos & derivados , Coagulación Sanguínea/efectos de los fármacos , Sinergismo Farmacológico , Factor Xa/metabolismo , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Trombina/metabolismoRESUMEN
RATIONALE: Mast cells and neutrophils are key contributors to the pathophysiological inflammatory processes that underpin asthma and chronic obstructive pulmonary disease, partly through the release of noxious serine proteases, including cathepsin G (Cat G) and chymase. From this standpoint, a dual inhibitor of neutrophil Cat G and mast cell chymase could protect against these disease-related inflammatory responses. OBJECTIVES: We examined the antiinflammatory pharmacology of RWJ-355871, a dual inhibitor of Cat G and chymase, in animal models of inflammation that evince pathophysiological pathways relevant to asthma and chronic obstructive pulmonary disease to determine the therapeutic potential of this compound. METHODS: In an ovalbumin (OVA)-sensitized rat model, RWJ-355871 was administered to block the mast-cell-mediated increase in paw volume caused by OVA injection. In a sheep asthma model, antigen-induced airway responses were assessed with and without aerosol treatment with RWJ-355871. In a murine tobacco-smoke model of airway inflammation, the effect of RWJ-355871 on smoke-induced neutrophilia was determined. MEASUREMENTS AND MAIN RESULTS: Intravenous treatment of OVA-sensitized rats with RWJ-355871 provided dose-dependent reduction in the increase in rat paw volume. In allergic sheep, aerosol pretreatment with RWJ-355871 showed dose-dependent inhibition of the antigen-induced early response, late response, and post-antigen-induced airway hyperreponsiveness. In tobacco-smoke-exposed mice, nebulized RWJ-355871 significantly reduced the smoke-induced neutrophilia from the levels observed in untreated mice. CONCLUSIONS: The preclinical antiinflammatory effects of RWJ-355871 in these animal models of inflammation indicate that this dual inhibitor may have therapeutic utility for treating airway inflammatory diseases involving mechanisms that depend on Cat G and/or chymase.
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Catepsina G/antagonistas & inhibidores , Quimasas/antagonistas & inhibidores , Enfermedades Pulmonares/enzimología , Organofosfonatos/uso terapéutico , Piperidinas/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Catepsina G/metabolismo , Quimasas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Inyecciones Intravenosas , Enfermedades Pulmonares/tratamiento farmacológico , Ratones , Organofosfonatos/administración & dosificación , Piperidinas/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Ratas , Ovinos , Resultado del TratamientoRESUMEN
Serine proteases such as thrombin, trypsin and mast cell tryptase can act on different cell types through protease-activated receptors (PARs). These receptors have been shown to be implicated in several phenomena such as inflammation, platelet activation, immune response and atherosclerosis. Several studies recently reported PARs expression on neurons and some of them demonstrated that these receptors could interfere with nociception. The contribution of PAR(1) to inflammatory pain and the mechanism involved in this phenomenon were investigated. Intraplantar injection of PAR(1) agonist increased withdrawal latency and reduced response frequency to von Frey filaments, thus inhibiting nociceptive response to both mechanical and thermal stimuli in mice. PAR(1) agonist also reduced carrageenan-induced inflammatory hyperalgesia. The anti-nociceptive effects of PAR(1) agonist were mediated by endogenous opioids, as this effect was inhibited by local injection of naloxone methiodide, and because intraplantar injection of PAR(1) agonist increased mRNA expression of the endogenous opioid precursor proenkephalin. However, PAR(1) agonist was not able to inhibit calcium signals in isolated sensory neurons exposed to pro-nociceptive agents. Finally, despite similar inflammatory parameters, PAR(1)-deficient mice showed a strong potentiation of inflammatory hyperalgesia induced by the intraplantar injection of either formalin or carrageenan, or in the chronic model of collagen-induced arthritis, compared to wild-type mice. This study highlights a previously unknown endogenous mechanism of analgesia, showing a central role for the thrombin receptor PAR(1) in the regulation of inflammatory pain and as an activator of opioid pathways.
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Inflamación/fisiopatología , Péptidos Opioides/fisiología , Dolor/fisiopatología , Receptores de Trombina/fisiología , Animales , Calcio/metabolismo , Pie , Hiperalgesia/inducido químicamente , Hiperalgesia/patología , Inmunohistoquímica , Inflamación/inducido químicamente , Inflamación/complicaciones , Inyecciones , Masculino , Ratones , Ratones Endogámicos C57BL , Nociceptores/efectos de los fármacos , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Dolor/inducido químicamente , Dolor/etiología , Dimensión del Dolor/efectos de los fármacos , Receptor PAR-1/agonistas , Receptor PAR-1/fisiología , Receptores Opioides/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Protease-activated receptor-2 (PAR(2)), a receptor highly expressed in the respiratory tract, can influence inflammation at mucosal surfaces. Although the effects of PAR(2) in the innate immune response to bacterial infection have been documented, knowledge of its role in the context of viral infection is lacking. We thus investigated the role of PAR(2) in influenza pathogenesis in vitro and in vivo. In vitro, stimulation of PAR(2) on epithelial cells inhibited influenza virus type A (IAV) replication through the production of IFN-gamma. In vivo, stimulation of PAR(2) using specific agonists protected mice from IAV-induced acute lung injury and death. This effect correlated with an increased clearance of IAV in the lungs associated with increased IFN- gamma production and a decreased presence of neutrophils and RANTES release in bronchoalveolar fluids. More importantly, the protective effect of the PAR(2) agonist was totally abrogated in IFN- gamma-deficient mice. Finally, compared with wild-type mice, PAR(2)-deficient mice were more susceptible to IAV infection and displayed more severe lung inflammation. In these mice higher neutrophil counts and increased RANTES concentration but decreased IFN- gamma levels were observed in the bronchoalveolar lavages. Collectively, these results showed that PAR(2) plays a protective role during IAV infection through IFN-gamma production and decreased excessive recruitment of inflammatory cells to lung alveoli.
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Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Interferón gamma/metabolismo , Receptor PAR-2/metabolismo , Animales , Línea Celular , Perros , Femenino , Regulación de la Expresión Génica , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Receptor PAR-2/agonistas , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Transducción de Señal , Tasa de Supervivencia , Regulación hacia ArribaRESUMEN
The G protein-coupled protease-activated receptors (PAR) are key signaling components for proteases in vascular biology and tumor progression. To address the contributions of PAR1 and PAR2 to breast cancer development, we established cohorts of mouse mammary tumor virus-polyoma middle T (PyMT) PAR1(-/-) and PAR2(-/-) mice, considering that the PyMT model recapitulates aspects of human disease. Appearance of palpable tumors, tumor expansion, and metastasis was indistinguishable between wild-type and PAR1(-/-) mice. PAR1(-/-) breast cancer cells were no longer responsive to thrombin in vitro, excluding compensatory up-regulation of alternative thrombin receptors and indicating that thrombin-PAR1 signaling is dispensable in breast tumor microenvironments. In contrast, palpable tumors and multifocal disease developed slower in PAR2(-/-) mice, and as a consequence of delayed tumor onset, metastasis was reduced. Analysis of early tumors showed persistence of adenomas with delayed appearance of vascularized adenocarcinomas in PAR2(-/-) mice. Furthermore, CXCL1 production by early PAR2(-/-) tumors was reduced. These results are consistent with previous xenograft data that implicated breast cancer PAR2 signaling in the induction of proangiogenic growth factors and chemokines. This study establishes that protease signaling contributes to mammary tumor development and that PAR2, rather than the thrombin receptor PAR1, plays a crucial role in the angiogenic switch.
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Adenocarcinoma/fisiopatología , Antígenos Transformadores de Poliomavirus/genética , Neoplasias Mamarias Experimentales/fisiopatología , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transducción de Señal , Adenocarcinoma/irrigación sanguínea , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Neoplasias Mamarias Experimentales/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Reacción en Cadena de la PolimerasaRESUMEN
The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.
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Antagonistas de los Receptores de Hormonas Antidiuréticas , Benzazepinas/farmacología , Compuestos de Espiro/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Hipertensión/tratamiento farmacológico , Técnicas In Vitro , Macaca fascicularis , Masculino , Agregación Plaquetaria/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Vasoconstricción , Vasopresinas/farmacologíaRESUMEN
Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.
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Coagulación Sanguínea/fisiología , Células Dendríticas/metabolismo , Inflamación/metabolismo , Receptor PAR-1/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Inflamación/inmunología , Sistema Linfático/inmunología , Ratones , Ratones Endogámicos C57BL , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor Cross-Talk/fisiología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Sepsis/inmunología , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors that are activated by endogenous serine proteinases that cleave the N-terminal domain of the receptor unmasking a "tethered ligand" sequence. Trypsin and other agonists at PAR(2) act on peripheral nerves to augment the transfer of nociceptive information. We tested whether PAR(2) agonists also exert a spinal pronociceptive effect by i.t. administering the selective ligand, Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLI-GRL). This produced thermal and mechanical hyperalgesia in rats and mice and augmented mechanical and thermal hyperalgesia seen in the formalin inflammatory pain test. Effects of SLIGRL were abrogated in PAR(2)-deficient mice and were not seen with the inactive control peptide, Leu-Arg-Gly-Ile-Leu-Ser-NH(2). Surprisingly, electrophysiological studies, using whole-cell recording from rat substantia gelatinosa neurons, failed to demonstrate an increase in excitatory transmission or neuronal excitability following treatment with SLIGRL or trypsin. In fact, the actions of trypsin were consistent with a decrease in dorsal horn excitability. SLIGRL and trypsin did, however, depolarize and increase the excitability of large, medium and small primary afferent, dorsal root ganglion neurons. The effects were associated with an increase in conductance at hyperpolarized potentials and a decrease in conductance at depolarized potentials. PAR(2)-like immunoreactivity was found in DRG but not in spinal dorsal horn. These results suggest that activation of DRG neuron cell bodies may account for the pronociceptive actions of i.t. applied PAR(2) agonists. They also imply that pathophysiological release of PAR(2)-activating proteases in the vicinity of DRG neurons may produce profound effects on nociceptive processing in vivo.
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Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Oligopéptidos/farmacología , Receptor PAR-2/agonistas , Animales , Formaldehído , Ganglios Espinales/fisiología , Calor , Hiperalgesia/inducido químicamente , Inyecciones Espinales , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Noqueados , Neuronas Aferentes/fisiología , Dolor/inducido químicamente , Dolor/fisiopatología , Ratas , Ratas Wistar , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Tripsina/farmacologíaRESUMEN
It has been demonstrated that trypsin is able to evoke the classical signals of inflammation, mainly via the activation of proteinase-activated receptor-2 (PAR-2). This study was designed to evaluate the inflammatory and nociceptive responses caused by trypsin injection in the mouse paw. Trypsin produced a dose- and time-related paw edema, a response that was markedly reduced in PAR-2-deficient mice compared to wild-type mice, particularly at the early time-points after trypsin injection. In addition, trypsin produced an increase in myeloperoxidase (MPO) activity, which was significantly reduced in PAR-2-deficient mice. The injection of trypsin into the mouse paw also elicited a dose- and time-dependent spontaneous nociception, as well as thermal and mechanical hypernociceptive responses, which were consistently decreased in mice with genetic deletion of PAR-2. Pharmacological evaluation revealed that edema formation and spontaneous nociception caused by trypsin injection in the mouse paw are mediated by a complex range of mediators. Both edema and nociception seem to rely on the production of neuropeptides, probably involving C-fibre activation and vanilloid receptor-1 (TRPV1), besides the stimulation of kinin B(2) receptors. Edematogenic response is also likely related to the production of cyclooxygenase (COX) metabolites, whereas the mast cell activation appears to be greatly associated to spontaneous nociception. Altogether, the present results indicate that trypsin-induced edema and nociception in the mouse paw represent multi-mediated responses that are largely, but not exclusively, related to the activation of PAR-2. These pieces of evidence provide new insights on the role of trypsin in pain and inflammation.
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Inflamación/inducido químicamente , Nociceptores/efectos de los fármacos , Dolor/fisiopatología , Tripsina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Masculino , Mastocitos/fisiología , Ratones , Nociceptores/fisiología , Receptor de Bradiquinina B2/fisiología , Receptor PAR-2/fisiología , Canales Catiónicos TRPV/fisiologíaRESUMEN
BACKGROUND: Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. METHODS AND RESULTS: We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. CONCLUSIONS: Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.
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Cardiomegalia/fisiopatología , Infarto del Miocardio/fisiopatología , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Remodelación Ventricular/fisiología , Animales , Cardiomegalia/diagnóstico por imagen , Cardiomiopatía Dilatada/diagnóstico por imagen , Cardiomiopatía Dilatada/fisiopatología , Ecocardiografía , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Infarto del Miocardio/diagnóstico por imagen , Miocitos Cardíacos/fisiología , Fenotipo , Daño por Reperfusión/fisiopatología , Tromboplastina/genética , Miosinas Ventriculares/genéticaRESUMEN
The proteinase-activated receptors (PARs) are a novel family of G protein-coupled receptors, and their effects in neurodegenerative diseases remain uncertain. Alzheimer's disease (AD) is a neurodegenerative disorder defined by misfolded protein accumulation with concurrent neuroinflammation and neuronal death. We report suppression of proteinase-activated receptor-2 (PAR2) expression in neurons of brains from AD patients, whereas PAR2 expression was increased in proximate glial cells, together with up-regulation of proinflammatory cytokines and chemokines and reduced IL-4 expression (p < 0.05). Glial PAR2 activation increased expression of formyl peptide receptor-2 (p < 0.01), a cognate receptor for a fibrillar 42-aa form of beta-amyloid (Abeta(1-42)), enhanced microglia-mediated proinflammatory responses, and suppressed astrocytic IL-4 expression, resulting in neuronal death (p < 0.05). Conversely, neuronal PAR2 activation protected human neurons against the toxic effects of Abeta(1-42) (p < 0.05), a key component of AD neuropathogenesis. Amyloid precursor protein-transgenic mice, displayed glial fibrillary acidic protein and IL-4 induction (p < 0.05) in the absence of proinflammatory gene up-regulation and neuronal injury, whereas PAR2 was up-regulated at this early stage of disease progression. PAR2-deficient mice, after hippocampal Abeta(1-42) implantation, exhibited enhanced IL-4 induction and less neuroinflammation (p < 0.05), together with improved neurobehavioral outcomes (p < 0.05). Thus, PAR2 exerted protective properties in neurons, but its activation in glia was pathogenic with secretion of neurotoxic factors and suppression of astrocytic anti-inflammatory mechanisms contributing to Abeta(1-42)-mediated neurodegeneration.
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Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/prevención & control , Receptor PAR-2/fisiología , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Animales , Astrocitos/metabolismo , Línea Celular , Células Cultivadas , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/toxicidad , Ratas , Receptor PAR-2/biosíntesis , Receptor PAR-2/deficiencia , Receptor PAR-2/genética , Receptores de Formil Péptido/biosíntesis , Receptores de Formil Péptido/genética , Receptores de Lipoxina/biosíntesis , Receptores de Lipoxina/genéticaRESUMEN
BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.