RESUMEN
The transcription factor NF-κB regulates the expression of multiple genes involved in inflammation, apoptotic cell death and cell survival. We previously demonstrated that activated protein C (APC), a serine protease of hemostasis with anticoagulant activity, protected cultured rat cortical and hippocampal neurons against glutamate-induced excitotoxicity, a model of ischemic stroke. We reported that APC suppressed the translocation of NF-κBp65/RelA into the nucleus of neurons. However, it is not known whether APC-induced protection of neurons against cell death occurs via regulation of NF-κB activation or NF-κB-independent p53 expression. It is also unclear whether cleaved caspase-3 and caspase-independent AIF and Bax/Bcl-2 expression are involved at excitotoxicity. To elucidate the NF-κB dependent and -independent mechanisms in the APC-mediated cell survival, we analyzed in cortical and hippocampal neurons the effects of helenalin, a specific inhibitor of NF-κB activity, and APC on neuronal cell death and on the level of nuclear AIF, p53, caspase-3 and the apoptotic index (Bax/Bcl-2 ratio). We could demonstrate that helenalin (5 µM), like APC (1 nM), protects cultured neurons from glutamate-induced excitotoxicity. Both APC and helenalin inhibit AIF release from mitochondria and its translocation into the nucleus. They decrease the apoptotic index in neurons at excitotoxicity. However, APC, but not helenalin, reduced the glutamate-induced activation of caspase-3. Incubation of neurons with APC blocked the glutamate-induced increase in the nuclear level of p53 via NF-κB-independent pathway. Our findings demonstrate that, in the protective effect of APC in neurons at excitotoxicity, the NF-κB pathway is an important, but not the only pathway, and is significantly connected with neuronal survival at excitotoxicity.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Hipocampo/efectos de los fármacos , FN-kappa B/metabolismo , Neuronas/efectos de los fármacos , Proteína C/farmacología , Animales , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Neuronas/metabolismo , Ratas , Ratas Wistar , Sesquiterpenos/farmacología , Sesquiterpenos de GuayanoRESUMEN
Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.
Asunto(s)
Corteza Cerebral/citología , Ácido Glutámico/toxicidad , Hipocampo/citología , Neuronas/efectos de los fármacos , Proteína C/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , L-Lactato Deshidrogenasa/metabolismo , Neuronas/metabolismo , Ratas , Ratas Wistar , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/genética , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Factores de TiempoRESUMEN
Thrombin receptor agonist peptide (TRAP-6) could advantageously replace thrombin in terms of accelerating wound healing being less expensive and more stable. To promote TRAP-6 pharmacological action as a tissue reconstruction stimulator this study investigated its entrapment within poly(D,L)-lactide-co-glycolide (PLGA) microparticles. Due to its low molecular weight and water solubility, TRAP-6 microencapsulated form is expected to be more useful. This paper reports TRAP-6 microencapsulation by a double (w/o/w) emulsion-evaporation technique. TRAP-6 release kinetics were evaluated by both chemical (HPLC) and biological assays in vitro. The results revealed a high level of TRAP-6 sensitivity to physico-chemical events during the microencapsulation. The surface morphology difference between control microparticles (without TRAP-6) and microparticles with entrapped TRAP-6 during in vitro degradation highlighted a particular role of TRAP-6. The results can allow one to optimize the microencapsulation procedure and to encounter a new promising approach to development of biodegradable polymer drug delivery systems for wound healing.
Asunto(s)
Fragmentos de Péptidos/química , Péptidos/química , Poliglactina 910 , Receptores de Trombina/agonistas , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Cápsulas , Preparaciones de Acción Retardada , Emulsiones , Humanos , Ácido Láctico/química , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Hemostatic serine proteinases-thrombin, Factor VIIa, Factor Xa, play the central role in blood coagulation and thrombosis. Activation of coagulation and generation of active proteinases is initiated by tissue factor (TF) that is expressed by cells of the innate immune system and endothelial cells after tissue damage and cell activation induced by trauma, infection, hypoxia and other cell injury. Coagulation and inflammation are the essential part of the defensive host response. These processes have several connecting points account for the associate and/or the interaction between coagulation and inflammation pathways. The first link between these processes is endothelium, which after damage expresses the adhesive proteins (vWF,P-selectin), inductors and receptors, involved in both coagulation and inflammation. The second link is platelets, which stored in and after activation release proteins with procoagulant and proinflammatory properties. The third link is the serine proteinases, which produced for blood coagulation and activate via its specifical receptors--PARs (proteinase activated receptors) the cells of both coagulation and inflammation system thereby controlling these processes. The generation of these proteinases is initiated by tissue factor (TF) which triggers blood coagulation at sites of tissue injury by selective binding of FVIIa. TF/VIIa complexes with substrate--FX that is activated to FXa. TF/VIIa/Xa can activate both the inflammatory responses of endothelial and other cells and also blood coagulation through stimulation of thrombin generation. This review summarizes the latest data on the blood coagulation activation that include generation of active surface for coagulation, generation of hemostatic serine proteinases and its role as signalling molecules that via PARs and other receptors involved in regulation and control of the interaction of blood coagulation and inflammation and illustrates the potential for therapeutic intervention.
Asunto(s)
Coagulación Sanguínea , Plaquetas/patología , Adhesión Celular , Péptido Hidrolasas/metabolismo , Receptores Proteinasa-Activados/metabolismo , Tromboplastina/metabolismo , Animales , Anticoagulantes/química , Plaquetas/inmunología , Endotelio Vascular/metabolismo , Factor VIIa/química , Factor Xa/química , Hemostasis , Humanos , Inflamación , Modelos Biológicos , Selectina-P/metabolismo , Unión Proteica , Serina Endopeptidasas/química , Transducción de Señal , Trombina/metabolismoRESUMEN
Functions of thrombin as a modulator of inflammation and tissue repair are mediated by the proteinase-activated receptor (PAR) family. Some of these effects may be induced by activation of mast cells. To characterize the degranulation of rat peritoneal mast cells in response to PAR agonists, the effects of thrombin, trypsin and peptide agonists of PARs (PAR-AP, proteinase-activated receptor-activating peptides) on secretion were investigated. The release of beta-hexosaminidase by thrombin (0.01-1 microM) was concentration-dependent and mediated via PAR(1), as evidenced by cathepsin G (100 microM)-induced inactivation of PAR(1) and thrombin-stimulated PAR(1) desensitization. Trypsin (1 microM) accelerated histamine secretion. The PAR(1)-AP, TRAP (SFFLRN, 1-100 microM) and the PAR(2)-AP SLIGRL (5-100 microM) caused the release of histamine, and beta-hexosaminidase from inflammatory mast cells were obtained from a model of acute peritonitis in rats. Relative to the response to compound 48/80, the thrombin- and TRAP-induced release of beta-hexosaminidase was higher in inflammatory mast cells than in the control. This suggests that additional exposure of PAR(1) on mast cells to PAR agonists or an increase in PARs sensitivity to PAR agonists probably occurred during acute inflammation.