RESUMEN
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.
Asunto(s)
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
BACKGROUND: We previously reported that the functional mutation of Toll-like receptor 4 (TLR4) in C3H/HeJ mice subjected to myocardial ischemia-reperfusion (MI/R) injury resulted in an attenuation of myocardial infarction size. To investigate the ligand-activating TLR4 during MI/R injury, we evaluated the effect of eritoran, a specific TLR4 antagonist, on MI/R injury, with the goal of defining better therapeutic options for MI/R injury. METHODS AND RESULTS: C57BL/6 mice received eritoran (5 mg/kg) intravenously 10 minutes before 30 minutes of in situ of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. Infarct size was measured using triphenyltetrazoliumchloride staining. A c-Jun NH(2)-terminal kinase (JNK) activation was determined by Western blotting, nuclear factor (NF)-kappaB activity was detected by gel-shift assay, and cytokine expression was measured by ribonuclease protection assay. Mice treated with eritoran developed significantly smaller infarcts when compared with mice treated with vehicle alone (21.0+/-6.4% versus 30.9+/-13.9%; P=0.041). Eritoran pretreatment resulted in a reduction in JNK phosphorylation (eritoran versus vehicle: 3.98+/-0.81 versus 7.01+/-2.21-fold increase; P=0.020), less nuclear NF-kappaB translocation (2.70+/-0.35 versus 7.75+/-0.60-fold increase; P=0.00007), and a decrease in cytokine expression (P<0.05). CONCLUSIONS: We conclude that inhibition of TLR4 with eritoran in an in situ murine model significantly reduces MI/R injury and markers of an inflammatory response.
Asunto(s)
Disacáridos/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Fosfatos de Azúcar/uso terapéutico , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Biomarcadores , Citocinas/biosíntesis , Citocinas/genética , Disacáridos/farmacología , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Inflamación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Fosfatos de Azúcar/farmacología , Receptor Toll-Like 4/fisiologíaRESUMEN
OBJECTIVES: p38 mitogen-activated protein kinase is associated with many clinical entities characterized by inflammation. We postulated that inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates inflammation and the development of pulmonary hypertension in monocrotaline-treated rats. METHODS: Rats were divided into 4 groups: (1) the control group (daily 0.9% saline), (2) the FR group (daily FR167653, 2 mg . kg(-1) . d(-1)), (3) the MCT group (daily 0.9% saline the day after a single monocrotaline dose, 60 mg/kg), and (4) the MCT+FR group (daily FR167653, 2 mg . kg(-1) . d(-1), the day after a single MCT dose). Body weight, pulmonary artery pressure, and morphometric changes of the pulmonary artery with the histopathologic method were observed weekly for 4 weeks. Also, p38 mitogen-activated protein kinase activity and inflammatory cytokine expression in the lung were measured. RESULTS: Four weeks after monocrotaline administration, mean pulmonary artery pressure in the MCT+FR group was lower than in the MCT group (MCT+FR vs MCT: 24.7 +/- 1.9 vs 36.5 +/- 2.1 mm Hg; P < .05). In morphometric analysis the percentage of medial wall thickness and the percentage of muscularization in the MCT+FR group were reduced compared with those in the MCT group after 4 weeks (P < .05); however, the number of macrophages was not significantly different. p38 mitogen-activated protein kinase activity was significantly attenuated in the MCT+FR group compared with in the MCT group (7.2 +/- 0.52 vs 2.1 +/- 0.23 fold-increase, P < .05, at 1 week). Although mRNA levels of tumor necrosis factor alpha and interleukin 1beta were reduced in the MCT+FR group compared with in the MCT group (tumor necrosis factor alpha: 1.18 +/- 0.36 vs 3.05 +/- 1.12 fold-increase, P < .05, at 2 weeks; interleukin 1beta: 2.2 +/- 0.34 vs 4.4 +/- 1.09 fold-increase, P < .05, at 1 week), FR167653 did not suppress increased monocyte chemotactic protein 1 mRNA expression induced by monocrotaline (3.2 +/- 0.62 vs 3.1 +/- 0.42 fold-increase, at 1 week). CONCLUSION: FR167653 significantly attenuates the expression of inflammatory cytokines, ultimately preventing the progression of pulmonary hypertension. These results suggest that p38 mitogen-activated protein kinase might play a central role in the molecular events that underlie the development and progression of pulmonary hypertension.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Hipertensión Pulmonar/fisiopatología , Pirazoles/farmacología , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Citocinas/metabolismo , Hipertensión Pulmonar/inducido químicamente , Hipertrofia Ventricular Derecha/fisiopatología , Inmunohistoquímica , Pulmón/citología , Masculino , Monocrotalina , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: During myocardial ischemia-reperfusion injury, p38 mitogen-activated protein kinase is activated. We examined the effect of a highly specific inhibitor of p38 mitogen-activated protein kinase, FR167653, in an experimental model of regional myocardial ischemia-reperfusion. METHODS: CD-1 mice received FR167653 intraperitoneally 24 hours before 30 minutes of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. The p38 mitogen-activated protein kinase activation and kinase activity were determined by Western blotting with monoclonal antibodies for the phosphorylated from of p38 mitogen-activated protein kinase or its substrate, activating transcription factor 2. Nuclear factor kappaB activity was measured by detecting translocation of nuclear factor kappaB to the nucleus. The expression of inflammatory cytokines was measured by ribonuclease protection assay. RESULTS: Pretreatment of mice with FR167653 before myocardial ischemia-reperfusion resulted in a reduction in p38 mitogen-activated protein kinase phosphorylation (P =.018), an inhibition of p38 mitogen-activated protein kinase activity (P =.047), a smaller amount of nuclear factor kappaB (P =.001), and a decrease in the expression of inflammatory cytokines (tumor necrosis factor alpha: P =.023, interleukin 1beta: P =.038, monocyte chemotactic protein 1: P =.0001) in the heart and the development of a significantly smaller infarct (P =.0069) relative to hearts from mice treated with vehicle alone. Activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase were observed after myocardial ischemia-reperfusion without inhibition by FR167653. CONCLUSION: We conclude that FR167653 selectively inhibits p38 mitogen-activated protein kinase activation and activity during regional myocardial ischemia-reperfusion injury and efficaciously reduces infarct size (by 73.6%). Thus p38 mitogen-activated protein kinase inhibition may have a role in the treatment of myocardial ischemia-reperfusion.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Animales , Western Blotting , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Premedicación , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND: Restoration of blood flow to the ischemic heart may paradoxically exacerbate tissue injury (ischemia/reperfusion injury). Toll-like receptor 4, expressed on several cell types, including cardiomyocytes, is a mediator of the host inflammatory response to infection. Because ischemia/reperfusion injury is characterized by an acute inflammatory reaction, we investigated toll-like receptor 4 activation in a murine model of regional myocardial ischemia/reperfusion injury. We used C3H/HeJ mice, which express a nonfunctional toll-like receptor 4, to assess the pertinence of this receptor to tissue injury after reperfusion of ischemic myocardium. METHODS: Wild-type mice (C3H/HeN) or toll-like receptor 4 mutant mice (C3H/HeJ) were subjected to 60 minutes of regional myocardial ischemia followed by 2 hours of reperfusion. At the end of reperfusion, the area at risk and the myocardial infarct size were measured as the end point of myocardial ischemia/reperfusion injury. Myocardial mitogen-activated protein kinase activation was measured by Western blotting, and nuclear translocation of nuclear factor-kappaB and activator protein-1 was determined by electrophoretic mobility shift assay. Ischemia/reperfusion-injured myocardium was also assessed by ribonuclease protection assay for expression of inflammatory mediators (tumor necrosis factor-alpha, interleukin-1beta, monocyte chemotactic factor-1, and interleukin-6). RESULTS: The area at risk was similar for all groups after myocardial ischemia/reperfusion injury. There was a 40% reduction in infarct size (as a percentage of the area at risk) in C3H/HeJ mice compared with C3H/HeN mice (P =.001). Within the myocardium, significant activation of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase was observed in both strains after ischemia and during reperfusion as compared with an absence of mitogen-activated protein kinase activation during sham operations; however, c-Jun N-terminal kinase activity, but not p38 or extracellular signal-regulated kinase activity, was significantly reduced in C3H/HeJ mice (P <.05). In both groups, nuclear factor-kappaB and activator protein-1 nuclear translocation occurred in the myocardium during myocardial ischemia/reperfusion injury, but, by densitometric analysis, nuclear translocation of nuclear factor-kappaB and activator protein-1 was significantly decreased in C3H/HeJ mice compared with C3H/HeN mice. Interleukin-1beta, monocyte chemotactic factor-1, and interleukin-6 were detectable in reperfused ischemic myocardium but were not detected in sham-operated myocardium; the expression of each of these mediators was significantly decreased in the myocardial tissue of C3H/HeJ mice when compared with expression in the control C3H/HeN mouse strain. CONCLUSIONS: Our data suggest that toll-like receptor 4 may mediate, at least in part, myocardial ischemia/reperfusion injury. Inhibition of toll-like receptor 4 activation may be a potential therapeutic target to attenuate ischemia/reperfusion-induced tissue damage in the clinical setting.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Daño por Reperfusión Miocárdica/etiología , Receptores de Superficie Celular/fisiología , Animales , Mediadores de Inflamación/fisiología , Ratones , Ratones Endogámicos C3H , Proteínas Quinasas Activadas por Mitógenos/fisiología , Infarto del Miocardio/etiología , Receptores Toll-LikeRESUMEN
We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(-/-)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(-/-) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(-/-) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Precondicionamiento Isquémico Miocárdico , Daño por Reperfusión Miocárdica/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Citoplasma/fisiología , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , ARN Mensajero/análisisRESUMEN
Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury.
Asunto(s)
Daño por Reperfusión Miocárdica/fisiopatología , Trombina/fisiología , Tromboplastina/fisiología , Ancrod/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Hirudinas/farmacología , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/patología , Miocardio/ultraestructura , ARN Mensajero/análisis , Conejos , Receptor PAR-1 , Receptores de Trombina/fisiologíaRESUMEN
Implantable ventricular assist devices have proven efficacious as a bridge to transplantation and as a bridge to recovery. Although current indications for use of assist devices are somewhat limited, they are likely to expand in the upcoming years, including their use as destination therapy for end-stage heart failure. Recipients of assist devices, however, are prone to certain device-specific complications, including excessive postoperative bleeding, late propensity for thromboembolism, infections, and systemic inflammation, which may contribute to end-organ dysfunction. This article reviews the systemic biochemical alterations underlying these clinical phenomena. As assist devices are increasingly used, better understanding of these systemic perturbations is imperative.