RESUMEN
Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/enzimología , Anciano , Animales , Femenino , Humanos , Inhibidores de la Lipooxigenasa/administración & dosificación , Masculino , Ratones , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Recovery from stroke is limited, in part, by an inhibitory environment in the postischemic brain, but factors preventing successful remodeling are not well known. Using cultured cortical neurons from mice, brain endothelial cells, and a mouse model of ischemic stroke, we show that signaling from the axon guidance molecule Sema3A via eicosanoid second messengers can contribute to this inhibitory environment. Either 90 nM recombinant Sema3A, or the 12/15-lipoxygenase (12/15-LOX) metabolites 12-HETE and 12-HPETE at 300 nM, block axon extension in neurons compared to solvent controls, and decrease tube formation in endothelial cells. The Sema3A effect is reversed by inhibiting 12/15-LOX, and neurons derived from 12/15-LOX-knockout mice are insensitive to Sema3A. Following middle cerebral artery occlusion to induce stroke in mice, immunohistochemistry shows both Sema3A and 12/15-LOX are increased in the cortex up to 2 wk. To determine whether a Sema3A-dependent damage pathway is activated following ischemia, we injected recombinant Sema3A into the striatum. Sema3A alone did not cause injury in normal brains. But when injected into postischemic brains, Sema3A increased cortical damage by 79%, and again, this effect was reversed by 12/15-LOX inhibition. Our findings suggest that blocking the semaphorin pathway should be investigated as a therapeutic strategy to improve stroke recovery.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Encéfalo/metabolismo , Semaforina-3A/metabolismo , Accidente Cerebrovascular/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Encéfalo/irrigación sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Inmunohistoquímica , Leucotrienos/metabolismo , Masculino , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Neuronas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Semaforina-3A/antagonistas & inhibidores , Semaforina-3A/genética , Transducción de Señal , Accidente Cerebrovascular/patologíaRESUMEN
Oxidative stress is a pathway of injury that is common to almost all neurological conditions. Hence, methods to scavenge radicals have been extensively tested for neuroprotection. However, saving neurons alone may not be sufficient in treating CNS disease. In this study, we tested the cytoprotective actions of the glutathione precursor gamma-glutamylcysteine ethyl ester (GCEE) in brain endothelium. First, oxidative stress was induced in a human brain microvascular endothelial cell line by exposure to H(2)O(2). Addition of GCEE significantly reduced formation of reactive oxygen species, restored glutathione levels which were reduced in the presence of H(2)O(2), and decreased cell death during H(2)O(2)-mediated injury. Next, we asked whether GCEE can also protect brain endothelial cells against oxygen-glucose deprivation (OGD). As expected, OGD disrupted mitochondrial membrane potentials. GCEE was able to ameliorate these mitochondrial effects. Concomitantly, GCEE significantly decreased endothelial cell death after OGD. Lastly, our in vivo experiments using a mouse model of brain trauma show that post-trauma (10 min after controlled cortical impact) administration of GCEE by intraperitoneal injection results in a decrease in acute blood-brain barrier permeability. These data suggest that the beneficial effects of GCEE on brain endothelial cells and microvessels may contribute to its potential efficacy as a neuroprotective agent in traumatic brain injury.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Lesiones Encefálicas/metabolismo , Permeabilidad Capilar/fisiología , Dipéptidos/uso terapéutico , Células Endoteliales/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Animales , Barrera Hematoencefálica/efectos de los fármacos , Lesiones Encefálicas/prevención & control , Permeabilidad Capilar/efectos de los fármacos , Línea Celular , Dipéptidos/farmacología , Células Endoteliales/efectos de los fármacos , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
12/15-Lipoxygenase (12/15-LOX) is an important mediator of brain injury following experimental stroke in rodents. It contributes to neuronal death, but the underlying mechanism remains unclear. We demonstrate here that in neuronal HT22 cells subjected to glutamate-induced oxidative stress, 12/15-LOX damages mitochondria, and this represents the committed step that condemns the cell to die. Importantly these events, including breakdown of the mitochondrial membrane potential, the production of reactive oxygen species, and cytochrome c release, can all be replicated by incubation of 12/15-LOX with mitochondria in vitro, without the need to add other cytosolic factors. Proteasome activity is required downstream of mitochondrial damage to complete the cell death cascade, but proteasome inhibition is only partially protective. These findings position 12/15-LOX as the central executioner in an oxidative stress-related neuronal death program.
Asunto(s)
Araquidonato 15-Lipooxigenasa/farmacología , Mitocondrias/efectos de los fármacos , Neuronas/ultraestructura , Estrés Oxidativo/fisiología , Animales , Células Clonales , Citocromos c/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Citometría de Flujo/métodos , Ácido Glutámico/farmacología , Glutatión/metabolismo , Hipocampo/citología , Inhibidores de la Lipooxigenasa/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Future demographic changes predict an increase in the number of patients with atrial fibrillation. As long-term anticoagulation for the prevention of ischemic strokes becomes more prevalent, the burden of warfarin-associated intracerebral hemorrhage (W-ICH) is likely to grow. However, little is known about the clinical aspects and pathophysiologic mechanisms of W-ICH. This study describes the development of a mouse model of W-ICH in which hematoma growth and outcomes can be correlated with anticoagulation parameters. METHODS: CD-1 mice were treated with warfarin (2 mg/kg per 24 hours) added to drinking water. ICH was induced by stereotactic injection of collagenase type VII (0.075 U) into the right striatum. Hemorrhagic blood volume was quantified by means of a photometric hemoglobin assay 2 and 24 hours after hemorrhage induction. Neurologic outcomes were assessed on a 5-point scale. RESULTS: The international normalized ratio in nonanticoagulated mice was 0.8+/-0.1. After 24 (W-24) and 30 (W-30) hours of warfarin pretreatment, international normalized ratio values increased to 3.5+/-0.9 and 7.2+/-3.4, respectively. Compared with nonanticoagulated mice, mean hemorrhagic blood volume determined 24 hours after hemorrhage induction was found to be 2.5-fold larger in W-24 mice (P=0.019) and 3.1-fold larger in W-30 mice (P<0.001, n=10 per group). Mortality at 24 hours after hemorrhage induction was 0% in nonanticoagulated mice, 10% in W-24 mice, and 30% in W-30 mice. Hematoma enlargement between 2 and 24 hours after hemorrhage induction was -1.4% for nonanticoagulated mice, 22.9% for W-24 mice, and 62.2% for W-30 mice. CONCLUSIONS: This study characterizes the first experimental model of W-ICH. It may be helpful in gaining further insights into the pathophysiology of W-ICH and may be used for testing the efficacy of treatment strategies, such as hemostatic therapy, in this severe subtype of stroke.
Asunto(s)
Anticoagulantes/toxicidad , Hemorragia Cerebral/inducido químicamente , Colagenasas/toxicidad , Modelos Animales de Enfermedad , Warfarina/toxicidad , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Daño Encefálico Crónico/etiología , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/fisiopatología , Colagenasas/administración & dosificación , Cuerpo Estriado , Progresión de la Enfermedad , Hematoma/inducido químicamente , Hematoma/fisiopatología , Inyecciones , Relación Normalizada Internacional , Masculino , Ratones , Colagenasa Microbiana , Trastornos del Movimiento/etiología , Warfarina/administración & dosificaciónRESUMEN
It was shown in previous work that the interaction of growth factors (GFs) with adenosine triphosphate (ATP) is essential for their neuroprotective effect. Here we investigated the nature of the association of human basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) with ATP. It was demonstrated that this interaction involves the formation of non-covalent ATP-GF complexes that are labile at low pH and that could be selectively purified and subjected to electrospray and MALDI-TOF mass spectrometry. The results obtained with these techniques indicated that the stability of the complexes is high. Main features of the procedure used here are: (1) reversed-phase purification of nucleotide-protein non-covalent complexes, (2) their detection with MALDI-TOF-MS using acid-free matrix, and/or (3) their measurement with ESI-MS using soft desolvation conditions. The methodology was successful in providing proof for the presence of various nucleotide-GF complexes. It was extended to other nucleotide-binding proteins (ribonuclease A) as well as proteins that do not exhibit nucleotide binding (lysozyme) as positive and negative control, respectively. Thus, the method demonstrated its general use for the investigation of a wide range of proteins interacting with nucleotides as long as their complexes are sufficiently stable to accommodate the experimental conditions.
Asunto(s)
Adenosina Trifosfato/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Adenosina Trifosfato/química , Sitios de Unión , Factor Neurotrófico Derivado del Encéfalo/química , Factor 2 de Crecimiento de Fibroblastos/química , Humanos , Factor de Crecimiento Nervioso/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
Fibroblast growth factor 2 (FGF2), an intensively studied heparin-binding cytokine, is an important modulator of cell growth and differentiation under both physiological and pathophysiological conditions. It has been shown recently that ATP binds to FGF2 and that this binding is crucial for its biological function. In this study we demonstrated that divalent cations were not necessary for binding of ATP to FGF2, but it could be demonstrated that heparin blocked the labelling of FGF2 with ATP indicating an involvement of the heparin-binding domain (aa 128-144) in ATP-binding. FGF2, bound to Heparin Sepharose, could be eluted with ATP and GTP, but not with cAMP, AMP or ADP. Successive mutation of positively charged amino acid residues located in the heparin-binding domain drastically reduced the signal intensity of [gamma-(32)P]ATP labelled FGF2 indicating that this domain is not only important for heparin binding to FGF2 but also for ATP-binding.
Asunto(s)
Adenosina Trifosfato/química , Factor 2 de Crecimiento de Fibroblastos/química , Heparina/química , Factor Apoptótico 1 Activador de Proteasas/química , Sitios de Unión , Diferenciación Celular , Proliferación Celular , Cromatografía/métodos , Proteínas HSP90 de Choque Térmico/química , Humanos , Modelos Estadísticos , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor known to cause cell proliferation, angiogenesis and neuroprotection. We have performed site-directed mutagenesis to identify the amino acids that are essential for heparin/growth factor interaction and for neuroprotection. Binding to heparin-acrylic beads was markedly reduced when lysine in position 134 of bFGF was replaced by alanine. Wildtype (wt)-bFGF was shown to protect rat primary cultures of embryonic hippocampal neurons against damage caused by staurosporine and to reduce the infarct size in mice after focal cerebral ischemia. These neuroprotective effects of wt-bFGF could not be shown for the mutant bFGF(K134A). Furthermore, phosphorylation of Akt and ERK1/2 was significantly reduced in cultured neurons treated with bFGF(K134A) indicating diminished intracellular signaling compared to neurons treated with wt-bFGF. In conclusion, lysine at position 134 of bFGF is essential for bFGF to bind heparin, then to interact with its receptor and, subsequently, to protect neurons against damage.
Asunto(s)
Infarto Encefálico/tratamiento farmacológico , Factor 2 de Crecimiento de Fibroblastos/química , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Lisina/química , Fármacos Neuroprotectores/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos/fisiología , Animales , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatología , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/química , Heparina/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/fisiopatología , Lisina/metabolismo , Masculino , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Proteína Oncogénica v-akt/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismoRESUMEN
The aim of this work was to test whether growth factors such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) undergo autophosphorylation and whether this affects their biological activity. Incubation of those growth factors with [gamma-(32)P]ATP resulted in phosphorylation in vitro. The phosphate bond was resistant to alkaline pH, yet acid-labile. Addition of alkaline phosphatase resulted in time and protein dependent dephosphorylation. Concomitantly, alkaline phosphatase abolished the neuroprotective effect of those growth factors upon oxygen and glucose deprivation and upon staurosporine-induced cell death. For those studies, we were using primary cultures of cortical and hippocampal neurons from embryonic and neonatal rats. Incubation of bFGF with non-hydrolyzable ATP-gammaS resulted in phosphorylation and in neuroprotection resistant to alkaline phosphatase. We conclude that bFGF, NGF and BDNF undergo autophosphorylation on site(s) other than serine, threonine, tyrosine and/or ATP-binding, and that this binding of phosphate is essential for neuroprotection in vivo.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
12/15-lipoxygenase (12/15-LOX) contributes to acute neuronal injury and edema formation in mouse models of middle cerebral artery occlusion (MCAO). The apoptosis-inducing factor (AIF) is implicated in caspase-independent forms of apoptosis, and has been linked to ischemic neuronal cell death. We show here that increased AIF in the peri-ischemic cortex of mouse colocalizes with 12/15-LOX after 2 h of MCAO. The 12/15-LOX inhibitor baicalein prevents the increase and nuclear localization of AIF, suggesting this pathway may be partially responsible for the neuroprotective qualities of baicalein. Using an established cell line model of neuronal oxidative stress, we show that 12/15-LOX activated after glutathione depletion leads to AIF translocation to the nucleus, which is abrogated by the 12/15-LOX inhibitor baicalein (control: 19.3%+/-6.8% versus Glutamate: 64.0%+/-8.2% versus glutamate plus baicalein: 11.4%+/-2.2%). Concomitantly, resident proteins of the ER are dispersed throughout the cell (control: 31.0%+/-8.4% versus glutamate: 70.0%+/-5.5% versus glutamate plus baicalein: 8.0%+/-2.7%), suggesting cell death through organelle damage. Taken together, these findings show that 12/15-LOX and AIF are sequential actors in a common cell death pathway that may contribute to stroke-induced brain damage.