RESUMEN
Neonatal arterial ischemic stroke is one of the more severe birth complications. The injury can result in extensive neurological damage and is robustly associated with later diagnoses of cerebral palsy (CP). An important part of efforts to develop new therapies include the on-going refinement and understanding of animal models that capture relevant clinical features of neonatal brain injury leading to CP. The potent vasoconstrictor peptide, Endothelin-1 (ET-1), has previously been utilised in animal models to reduce local blood flow to levels that mimic ischemic stroke. Our previous work in this area has shown that it is an effective and technically simple approach for modelling ischemic injury at very early neonatal ages, resulting in stable deficits in motor function. Here, we aimed to extend this model to also examine the impact on cognitive function. We show that focal delivery of ET-1 to the cortex of Sprague Dawley rats on postnatal day 0 (P0) resulted in impaired learning in a touchscreen-based test of visual discrimination and correlated with important clinical features of CP including damage to large white matter structures.
Asunto(s)
Isquemia Encefálica/complicaciones , Parálisis Cerebral/etiología , Modelos Animales de Enfermedad , Endotelina-1/toxicidad , Vasoconstrictores/toxicidad , Animales , Animales Recién Nacidos , Aprendizaje por Asociación , Atrofia , Isquemia Encefálica/inducido químicamente , Isquemia Encefálica/patología , Recuento de Células , Corteza Cerebral/patología , Parálisis Cerebral/patología , Trastornos del Conocimiento/etiología , Cuerpo Estriado/patología , Endotelina-1/administración & dosificación , Inflamación , Inyecciones , Microglía/patología , Trastornos del Movimiento/etiología , Neuronas/patología , Trastornos de la Percepción/etiología , Ratas , Ratas Sprague-Dawley , Prueba de Desempeño de Rotación con Aceleración Constante , Vasoconstrictores/administración & dosificación , Sustancia Blanca/patologíaRESUMEN
OBJECTIVE: To investigate whether intravenously infused provokes migraine aura and migraine headache in migraine patients with aura. BACKGROUND: Migraine with aura has been associated with endothelial dysfunction and increased stroke risk. The initiating mechanism of migraine aura symptoms is not known. Experimental provocation of migraine headache using vasoactive peptides has provided tremendous advances in the understanding of migraine pathophysiology but substances that can induce migraine aura have not been identified. Endothelin-1 (ET-1), an endogenous, potent vasoconstrictor peptide released from the vascular endothelium, has been proposed to trigger migraine aura. This hypothesis is based on reports of increased plasma ET-1 levels early during the migraine attacks and the observation that ET-1 applied to the cortical surface potently induces the cortical spreading depolarization, the underlying electrophysiological phenomenon of migraine aura, in animals. Further, endothelial damage due to, for example, carotid puncture and vascular pathology is known to trigger aura episodes. METHODS: We investigated whether intravascular ET-1 would provoke migraine aura in patients. Using a two-way crossover, randomized, placebo-controlled, double-blind design, we infused high-dose (8 ng/kg/minutes for 20 minutes) intravenous ET-1 in patients with migraine with typical aura. The primary end-point was the difference in incidence of migraine aura between ET-1 and placebo. Experiments were carried out at a public tertiary headache center (Danish Headache Center, Rigshospitalet Glostrup, Denmark). RESULTS: Fourteen patients received intravenous ET-1. No patients reported migraine aura symptoms or migraine headache during or up to 24 hours following the ET-1 infusion. Four patients reported mild to moderate headache only on the ET-1 day, 3 patients reported moderate headache on the placebo day, and 1 patient reported mild headache on both days. No serious adverse events occurred during or after infusion. CONCLUSIONS: Provocation of migraine aura by procedures or conditions involving vascular irritation is unlikely to be mediated by ET-1.
Asunto(s)
Endotelina-1/farmacología , Trastornos Migrañosos/inducido químicamente , Adolescente , Adulto , Estudios Cruzados , Método Doble Ciego , Endotelina-1/administración & dosificación , Femenino , Humanos , Infusiones Intravenosas , Masculino , Migraña con Aura/inducido químicamente , Adulto JovenRESUMEN
Post-stroke depression (PSD) and post-stroke anxiety (PSA) are usually undertreated and many cases may remain undiagnosed, indicating a need for a better understanding of the underlying mechanisms. Current animal models of PSD and PSA using the middle cerebral artery occlusion model may be associated with motor deficits that can interfere with behavioral tests of depression- and anxiety-like behavior. Unilateral lesions of the medial prefrontal cortex (mPFC) have been reported to induce a depression- and anxiety-like phenotype in mice. The aim of this study was to examine the effects of unilateral microinjections of the vasoconstrictor endothelin-1 (ET-1) in the mPFC alone or in combination with the nucleus accumbens (NAc) on the behavior of rats after 2 and 6 weeks. Specifically, we measured anxiety- and depressive-like behavior, locomotion, and cognition. ET-1 injections in the mPFC and NAc resulted in replicable and localized lesions. Lesions to the mPFC and NAc resulted in more time spent in the open arms of the Elevated Plus Maze compared to sham-operated animals at 2 weeks post stroke, indicating decreased anxiety. This effect did not persist until 6 weeks post injection. No differences in locomotion, cognition and depressive-like behavior were found at either time point. In summary, unilateral lesions of mPFC and NAc did not produce a reliable and persistent anxiety and depression phenotype in rats.
Asunto(s)
Ansiedad/fisiopatología , Conducta Animal/fisiología , Cognición/fisiología , Locomoción/fisiología , Núcleo Accumbens/patología , Núcleo Accumbens/fisiopatología , Corteza Prefrontal/patología , Corteza Prefrontal/fisiopatología , Animales , Isquemia Encefálica/inducido químicamente , Isquemia Encefálica/patología , Depresión , Modelos Animales de Enfermedad , Endotelina-1/administración & dosificación , Masculino , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/administración & dosificaciónRESUMEN
Ischemic stroke accounts for 70-80% of stroke cases worldwide and survivors are frequently left with compromising sensorimotor deficits localized to one or more body regions. Most animal models of stroke involve transient or permanent occlusion of one or more major vessels such as the middle cerebral artery and are characterized by widespread damage to cortical and subcortical structures that result in deficits that can confound studies of neuroprotection and neurorehabilitation. Localized microinjections of the vasoconstricting peptide endothelin-1 (ET-1) into specific brain regions are becoming increasingly popular for such studies, but the pharmacology of endothelin-induced ischemic damage is poorly understood. To test the hypothesis that NMDA receptors, and particularly those containing the NR2B subunit, are involved in ET-1-mediated excitotoxicity and functional impairment, male CD1 rats (N = 32) were pre-treated with either the non-competitive NMDA antagonist MK-801 or the NR2B-selective antagonist Ro25-6981 (or vehicle) prior to unilateral microinjections of endothelin-1 into the somatosensory cortex and striatum. Rats were then tested using 4 established tests of sensory and/or motor function over 14 days. Lesion volumes were quantified post-mortem using standard histology and image analysis. Results confirmed reproducible lesions and significant deficits in all tests in vehicle-treated rats that were significantly reduced in both drug groups but were not different between drugs, providing evidence that endothelin-induced ischemic damage is mediated almost exclusively by NR2B-containing NMDA receptors.
Asunto(s)
Isquemia Encefálica/inducido químicamente , Endotelina-1/toxicidad , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Isquemia Encefálica/fisiopatología , Maleato de Dizocilpina/administración & dosificación , Endotelina-1/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Miembro Anterior/efectos de los fármacos , Miembro Anterior/fisiología , Masculino , Microinyecciones/métodos , Fenoles/administración & dosificación , Piperidinas/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
Large cerebral infarctions are major predictors of death and severe disability from stroke. Conversely, data concerning these types of infarctions and the affected adjacent brain circuits are scarce. It remains to be determined if the co-morbid concurrence of large infarct and ß-amyloid (Aß) toxicity can precipitate the early development of dementia. Here, we described a dose-dependent effect of a unilateral striatal injection of vasoconstrictive endothelin-1 (ET-1) along with Aß toxicity on CNS pathogenesis; driven by the anatomical and functional networks within a brain circuit. After 21 days of treatment, a high dose (60 pmol) of ET-1 (E60) alone caused the greatest increase in neuroinflammation, mainly in the ipsilateral striatum and distant regions with synaptic links to the striatal lesion such as white matter (subcortical white matter, corpus callosum, internal capsule, anterior commissure), gray matter (globus pallidus, thalamus), and cortices (cingulate, motor, somatosensory, entorhinal). The combined E60 + Aß treatment also extended perturbation in the contralateral hemisphere of these rats, such as increased deposition of amyloid precursor protein fragments associated with the appearance of degenerating cells and the leakage of laminin from the basement membrane across a compromised blood-brain barrier. However, the cerebral damage induced by the 6 pmol ET-1 (E6), Aß and E6 + Aß rats was not detrimental enough to injure the complete network. The appreciation of the causal interactions among distinct anatomical units in the brain after ischemia and Aß toxicity will help in the design of effective and alternative therapeutics that may disassociate the synergistic or additive association between the infarcts and Aß toxicity.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Encéfalo/patología , Infarto Cerebral/patología , Endotelina-1/toxicidad , Red Nerviosa/patología , Péptidos beta-Amiloides/administración & dosificación , Animales , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Infarto Cerebral/inducido químicamente , Endotelina-1/administración & dosificación , Inyecciones Intraventriculares , Masculino , Red Nerviosa/anatomía & histología , Red Nerviosa/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Endothelin-1 (ET-1) is a highly potent vasoconstrictor peptide released from vascular endothelium. ET-1 plays a major role in cerebrovascular disorders and likely worsens the outcome of acute ischaemic stroke and aneurismal subarachnoid haemorrhage through vasoconstriction and cerebral blood flow (CBF) reduction. Disorders that increase the risk of stroke, including hypertension, diabetes mellitus, and acute myocardial infarction, are associated with increased plasma levels of ET-1. The in vivo human cerebrovascular effects of systemic ET-1 infusion have not previously been investigated. In a two-way crossover, randomized, double-blind design, we used advanced 3 tesla MRI methods to investigate the effects of high-dose intravenous ET-1 on intra- and extracranial artery circumferences, global and regional CBF, and cerebral metabolic rate of oxygen (CMRO2) in 14 healthy volunteers. Following ET-1 infusion, we observed a 14% increase of mean arterial blood pressure, a 5% decrease of middle cerebral artery (MCA) circumference, but no effects on extracerebral arteries and no effects on CBF or CMRO2. Collectively, the findings indicate MCA constriction secondarily to blood pressure increase and not due to a direct vasoconstrictor effect of ET-1. We suggest that, as opposed to ET-1 in the subarachnoid space, intravascular ET-1 does not exert direct cerebrovascular effects in humans.
Asunto(s)
Encéfalo/diagnóstico por imagen , Circulación Cerebrovascular/efectos de los fármacos , Endotelina-1/farmacología , Imagen por Resonancia Magnética/métodos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Adulto , Encéfalo/irrigación sanguínea , Angiografía Cerebral/métodos , Endotelina-1/administración & dosificación , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/efectos de los fármacos , Femenino , Voluntarios Sanos , Humanos , Infusiones Intravenosas , Masculino , Arteria Cerebral Media/diagnóstico por imagen , Arteria Cerebral Media/efectos de los fármacos , Marcadores de SpinRESUMEN
Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor-α (TNF-α). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF-α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Endotelina-1/administración & dosificación , Endotelina-1/farmacología , Serina Endopeptidasas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Técnicas de Inactivación de Genes , Hemodinámica/efectos de los fármacos , Inyecciones Espinales , Interleucina-33/deficiencia , Interleucina-33/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.
Asunto(s)
Isquemia Encefálica/inducido químicamente , Caspasa 3/metabolismo , Corteza Cerebral/efectos de los fármacos , Endotelina-1/toxicidad , Animales , Isquemia Encefálica/patología , Caspasa 3/genética , Muerte Celular/efectos de los fármacos , Corteza Cerebral/lesiones , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Endotelina-1/administración & dosificación , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patologíaRESUMEN
Clearance of particles from the knee is an essential mechanism to maintain healthy joint homeostasis and critical to the delivery of drugs and therapeutics. One of the limitations in developing disease modifying drugs for joint diseases, such as osteoarthritis (OA), has been poor local retention of the drugs. Enhancing drug retention within the joint has been a target of biomaterial development, however, a fundamental understanding of joint clearance pathways has not been characterized. We applied near-infrared (NIR) imaging techniques to assess size-dependent in vivo clearance mechanisms of intra-articular injected, fluorescently-labelled polyethylene glycol (PEG-NIR) conjugates. The clearance of 2â¯kDa PEG-NIR (τâ¯=â¯171⯱â¯11â¯min) was faster than 40â¯kDa PEG-NIR (τâ¯=â¯243⯱â¯16â¯min). 40â¯kDa PEG-NIR signal was found in lumbar lymph node while 2â¯kDa PEG-NIR signal was not. Thus, these two conjugates may be cleared through different pathways, i.e. lymphatics for 40â¯kDa PEG-NIR and venous for 2â¯kDa PEG-NIR. Endothelin-1 (ET-1), a potent vasoconstrictor of vessels, is elevated in synovial fluid of OA patients but, its effects on joint clearance are unknown. Intra-articular injection of ET-1 dose-dependently inhibited the clearance of both 2â¯kDa and 40â¯kDa PEG-NIR. ET-1 caused a 1.63⯱â¯0.17-fold increase in peak fluorescence for 2â¯kDa PEG-NIR and a 1.85⯱â¯0.15-fold increase for 40â¯kDa PEG-NIR; and ET-1 doubled their clearance time constants. The effects of ET-1 were blocked by co-injection of ET receptor antagonists, bosentan or BQ-123. These findings provide fundamental insight into retention and clearance mechanisms that should be considered in the development and delivery of drugs and biomaterial carriers for joint diseases. STATEMENT OF SIGNIFICANCE: This study demonstrates that in vivo knee clearance can be measured using NIR technology and that key factors, such as size of materials and biologics, can be investigated to define joint clearance mechanisms. Therapies targeting regulation of joint clearance may be an approach to treat joint diseases like osteoarthritis. Additionally, in vivo functional assessment of clearance may be used as diagnostics to monitor progression of joint diseases.
Asunto(s)
Materiales Biocompatibles/química , Portadores de Fármacos/química , Endotelina-1/química , Articulación de la Rodilla/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Polietilenglicoles/química , Animales , Bosentán/química , Bosentán/farmacología , Liberación de Fármacos , Endotelina-1/administración & dosificación , Colorantes Fluorescentes/química , Inyecciones Intraarticulares , Cinética , Masculino , Imagen Óptica , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Líquido Sinovial/efectos de los fármacos , Distribución TisularRESUMEN
Emerging evidence indicates that endogenous production of endothelin (ET)-1, a 21-amino acid peptide vasoconstrictor, plays an important role in proteinuric kidney disease. Previous studies in rats have shown that chronic administration of ET-1 leads to increased glomerular albumin leakage. The underlying mechanisms are, however, currently not known. Here, we used size-exclusion chromatography to measure glomerular sieving coefficients for neutral FITC-Ficoll (molecular Stokes-Einstein radius: 15-80 Å, molecular weight: 70 kDa/400 kDa) in anesthetized male Sprague-Dawley rats (n = 12) at baseline and at 5, 15, 30, and 60 min after intravenous administration of ET-1. In separate experiments, ET-1 was given together with the selective ET type A (ETA) or ET type B (ETB) receptor antagonists JKC-301 and BQ-788, respectively. At both 15 and 30 min postadministration, the glomerular sieving coefficient for macromolecular Ficoll (70 Å) was significantly increased to 4.4 × 10-5 ± 0.7 × 10-5 (P = 0.024) and 4.5 × 10-5 ± 0.8 × 10-5 (P = 0.007), respectively, compared with baseline (2.2 × 10-5 ± 0.4 ×10-5). Decreased urine production after ET-1 prevented the use of higher doses of ET-1. Data analysis using the two-pore model indicated changes in large-pore permeability after ET-1, with no changes in the small-pore pathway. Administration of ETA blocker abrogated the permeability changes induced by ET-1 at 30 min, whereas blockade of ETB receptors was ineffective. Mean arterial pressure was only significantly increased at 60 min, being 123 ± 4 mmHg compared with 111 ± 2 mmHg at baseline (P = 0.02). We conclude that ET-1 evoked small, delayed, and sustained increases in glomerular permeability, mediated via the ETA receptor.
Asunto(s)
Endotelina-1/administración & dosificación , Tasa de Filtración Glomerular/efectos de los fármacos , Glomérulos Renales/efectos de los fármacos , Receptor de Endotelina A/agonistas , Animales , Hemodinámica/efectos de los fármacos , Infusiones Intravenosas , Glomérulos Renales/metabolismo , Masculino , Modelos Biológicos , Permeabilidad , Ratas Sprague-Dawley , Receptor de Endotelina A/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Endothelin-1 (ET-1), as it functions as a neuromodulator, has been associated with hypertension in chronic intermittent hypoxia (CIH) which attribute to enhanced carotid body sensibility to hypoxia. However, the molecular mechanism of ET-1 on carotid body sensibility in CIH is still not clear. Here, effect of ET-1 on carotid body chemosensory stimulation in rats exposed to either CIH or room air (Normoxia) was explored. Furthermore, Phospholipase C (PLC), Protein kinase C (PKC) or p38 MAPK antagonists were adopted to clarify the signalling pathways involved. Results showed that ET-1 induced a higher increase of carotid sinus nerve activity (CSNA) in animals exposed to CIH. Both ETA and ETB receptor expression were up-regulated by CIH exposure, but only ETA is responsible for ET-1 induced CSNA increase. Additional, the increase was inhibited by PLC, PKC, p38 MAPK antagonists and calcium channel blocker. Our findings support that ETA receptor mediates ET-1-induced CSNA increase through PLC, PKC and p38 MAPK signalling pathways in chronic intermittent hypoxia. Also, our study indicated that calcium influx was necessary for enhancing effect of ET-1 on CSNA.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Endotelina-1/metabolismo , Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores de Endotelina/metabolismo , Animales , Arterias Carótidas/inervación , Arterias Carótidas/fisiopatología , Cuerpo Carotídeo/efectos de los fármacos , Endotelina-1/administración & dosificación , Masculino , Proteína Quinasa C/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cerebral palsy is an irreversible movement disorder resulting from cerebral damage sustained during prenatal or neonatal brain development. As survival outcomes for preterm injury improve, there is increasing need to model ischemic injury at earlier neonatal time-points to better understand the subsequent pathological consequences. Here we demonstrate a novel neonatal ischemic model using focal administration of the potent vasoconstrictor peptide, endothelin-1 (ET-1), in newborn rats. The functional and histopathological outcomes compare favourably to those reported following the widely used hypoxic ischemia (HI) model. These include a robust motor deficit sustained into adulthood and recapitulation of hallmark features of preterm human brain injury, including atrophy of subcortical white matter and periventricular fiber bundles. Compared to procedures involving carotid artery manipulation and periods of hypoxia, the ET-1 ischemia model represents a rapid and technically simplified model more amenable to larger cohorts and with the potential to direct the locus of ischemic damage to specific brain areas.
Asunto(s)
Lesiones Encefálicas/patología , Encéfalo/patología , Endotelina-1/farmacología , Hipoxia-Isquemia Encefálica/patología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Lesiones Encefálicas/tratamiento farmacológico , Modelos Animales de Enfermedad , Endotelina-1/administración & dosificación , Femenino , Hipoxia/patología , Hipoxia-Isquemia Encefálica/inducido químicamente , Embarazo , RatasRESUMEN
Research into stroke is driven by frustration over the limited available therapeutics. Targeting a single aspect of this multifactorial disease contributes to the therapeutic boundaries. To overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding excitatory amino acid transporter 2 (EAAT2(, glutamate dehydrogenase 2 (GDH2), and nuclear factor E2-related factor 2 (Nrf2) genes, that acts synergistically to address the effected excito-oxidative axis. Here, we used the vasoconstrictor endothelin-1 (ET-1) to induce focal ischemic injury in mice by direct injection into the striatum. Mice treated with the mixture of these three genes show significant improvement in body balance, motor coordination, and decreased motor asymmetry compared to each gene separately. These results demonstrate that overexpression of the combined EAAT2, GDH2, and NRF2 genes can provide neuroprotection after ischemic injury.
Asunto(s)
Isquemia Encefálica/terapia , Terapia Genética/métodos , Accidente Cerebrovascular/terapia , Animales , Isquemia Encefálica/etiología , Endotelina-1/administración & dosificación , Endotelina-1/toxicidad , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Accidente Cerebrovascular/etiologíaRESUMEN
PURPOSE: Whilst the pathogenesis of retinal vein occlusion (RVO) is still unclear, systemic hypertension and increased level of endothelin-1 (ET-1) are known risk factors. Therefore, we studied the influence of ET-1 on the retinal veins in hypertensive rats. METHODS: We focused on the behavior of retinal veins in spontaneous hypertensive rats (SHR). To determine whether ET-1 was associated with the blood flow in eyes of SHRs, the chorioretinal blood flow in the rats was assessed using laser speckle flowgraphy (LSFG-Micro, Softcare, Fukuoka, Japan) before and after an intravenous injection of ET-1 under general anesthesia. In addition, retinas from SHRs and age-matched normotensive Wistar-Kyoto rats (WKYs) were removed, and retinal sections were immunostained for the ET-A and ET-B receptors. The protein levels of both ET-1 receptors and hypoxia-inducible factor 1 (HIF-1) in the retinal tissues were also determined by western blot analysis. RESULTS: One of the retinal veins became exceptionally constricted and was nearly occluded, and the chorioretinal blood flow significantly decreased in the retinas of SHRs following the injection of ET-1. Immunoreactivity to ET-A receptor was higher in SHR retinas than in WKY retinas. The protein levels of ET-A receptor and HIF-1 were also significantly higher in SHR retinas than in WKY retinas. CONCLUSIONS: An increase of ET-1 in circulating blood leads to the local constriction of retinal veins and this effect is accentuated in hypertensive rats by an upregulation of ET-A receptor. It is plausible that such a constriction of retinal veins increases retinal venous pressure, and may even contribute to the pathogenesis of RVO.
Asunto(s)
Endotelina-1/administración & dosificación , Oclusión de la Vena Retiniana/etiología , Oclusión de la Vena Retiniana/fisiopatología , Vena Retiniana/fisiopatología , Animales , Coroides/irrigación sanguínea , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inyecciones Intravenosas , Flujometría por Láser-Doppler , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Flujo Sanguíneo Regional/fisiología , Oclusión de la Vena Retiniana/metabolismo , Vasos Retinianos/fisiología , VasoconstricciónRESUMEN
Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.
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Endotelina-1/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Dolor/inducido químicamente , Dolor/metabolismo , Tacto/fisiología , Animales , Endotelina-1/administración & dosificación , Endotelina-1/toxicidad , Femenino , Inyecciones Subcutáneas , Masculino , Factor de Crecimiento Nervioso/administración & dosificación , Factor de Crecimiento Nervioso/toxicidad , Dimensión del Dolor/métodos , Estimulación Física/efectos adversos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Tacto/efectos de los fármacosRESUMEN
Excessive production of endothelin-1 (ET-1) has been observed in almost all forms of pulmonary hypertension. ET-1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pHi ), typically due to activation of Na+ /H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET-1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET-1-induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH-sensitive dye, BCECF-AM, to measure changes in resting pHi and NHE activity induced by exposure to exogenous ET-1 (10-8 mol/L) for 24 h. Cell migration and proliferation in response to ET-1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET-1 had no effect on NHE1 expression, but increased pHi , NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET-1-induced changes in cell function (proliferation and migration). Our results indicate that ET-1 modulates PASMC migration and proliferation via changes in pHi homeostasis through a pathway involving ROCK.
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Movimiento Celular , Proliferación Celular , Endotelina-1/metabolismo , Músculo Liso Vascular/metabolismo , Arteria Pulmonar/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Endotelina-1/administración & dosificación , Concentración de Iones de Hidrógeno , Masculino , Músculo Liso Vascular/efectos de los fármacos , Cultivo Primario de Células , Arteria Pulmonar/efectos de los fármacos , Ratas WistarRESUMEN
In utero hypoxia influences the structure and function of most fetal arteries, including those of the developing cerebral circulation. Whereas the signals that initiate this hypoxic remodeling remain uncertain, these appear to be distinct from the mechanisms that maintain the remodeled vascular state. The present study explores the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1), a potent vascular contractant and mitogen. In fetal lambs, chronic hypoxia (3,820-m altitude for the last 110 days of gestation) had no significant effect on plasma ET-1 levels or ETA receptor density in cerebral arteries but enhanced contractile responses to ET-1 in an ETA-dependent manner. In organ culture (24 h), 10 nM ET-1 increased medial thicknesses less in hypoxic than in normoxic arteries, and these increases were ablated by inhibition of PKC (chelerythrine) in both normoxic and hypoxic arteries but were attenuated by inhibition of CaMKII (KN93) and p38 (SB203580) in normoxic but not hypoxic arteries. As indicated by Ki-67 immunostaining, ET-1 increased medial thicknesses via hypertrophy. Measurements of colocalization between MLCK and SMαA revealed that organ culture with ET-1 also promoted contractile dedifferentiation in normoxic, but not hypoxic, arteries through mechanisms attenuated by inhibitors of PKC, CaMKII, and p38. These results support the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to ET-1 through pathways dependent upon PKC, CaMKII, and p38 that cause increased ET-1-mediated contractility, decreased ET-1-mediated smooth muscle hypertrophy, and a depressed ability of ET-1 to promote contractile dedifferentiation.
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Diferenciación Celular/genética , Arterias Cerebrales/metabolismo , Endotelina-1/genética , Hipoxia/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Arterias Cerebrales/embriología , Endotelina-1/administración & dosificación , Endotelina-1/sangre , Femenino , Feto/irrigación sanguínea , Feto/metabolismo , Hipoxia/sangre , Hipoxia/fisiopatología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Técnicas de Cultivo de Órganos , Embarazo , Proteína Quinasa C/genética , Ovinos , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/genética , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Endothelin (ET) receptor antagonists are potentially novel therapeutic agents in chronic kidney disease and resistant hypertension, but their use is complicated by sodium and water retention. In animal studies, this side effect arises from ETB receptor blockade in the renal tubule. Previous attempts to determine whether this mechanism operates in humans have been confounded by the hemodynamic consequences of ET receptor stimulation/blockade. We aimed to determine the effects of ET signaling on salt transport in the human nephron by administering subpressor doses of the ET-1 precursor, big ET-1. We conducted a 2-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. After sodium restriction, subjects received either intravenous placebo or big ET-1, in escalating dose (≤300 pmol/min). This increased plasma concentration and urinary excretion of ET-1. Big ET-1 reduced heart rate (≈8 beats/min) but did not otherwise affect systemic hemodynamics or glomerular filtration rate. Big ET-1 increased the fractional excretion of sodium (from 0.5 to 1.0%). It also increased free water clearance and tended to increase the abundance of the sodium-potassium-chloride cotransporter (NKCC2) in urinary extracellular vesicles. Our protocol induced modest increases in circulating and urinary ET-1. Sodium and water excretion increased in the absence of significant hemodynamic perturbation, supporting a direct action of ET-1 on the renal tubule. Our data also suggest that sodium reabsorption is stimulated by ET-1 in the thick ascending limb and suppressed in the distal renal tubule. Fluid retention associated with ET receptor antagonist therapy may be circumvented by coprescribing potassium-sparing diuretics.
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Endotelina-1 , Insuficiencia Renal Crónica , Sodio/metabolismo , Adulto , Animales , Diuresis/efectos de los fármacos , Diuresis/fisiología , Método Doble Ciego , Antagonistas de los Receptores de Endotelina/administración & dosificación , Antagonistas de los Receptores de Endotelina/efectos adversos , Antagonistas de los Receptores de Endotelina/farmacocinética , Endotelina-1/administración & dosificación , Endotelina-1/efectos adversos , Endotelina-1/farmacocinética , Femenino , Tasa de Filtración Glomerular , Humanos , Túbulos Renales/metabolismo , Túbulos Renales/fisiopatología , Masculino , Natriuresis/efectos de los fármacos , Natriuresis/fisiología , Receptores de Endotelina/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Resultado del Tratamiento , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Endothelin receptor A (ETAR) promotes tumorigenesis by stimulating cell proliferation, migration, and survival. However, the mechanism of ETAR in promoting tumor growth is largely unknown. In this study, we demonstrate that ETAR stimulates colon cell proliferation, migration, and tumorigenesis through the activation of YAP/TAZ, two transcription coactivators of the Hippo tumor suppressor pathway. Endothelin-1 treatment induced YAP/TAZ dephosphorylation, nuclear accumulation, and transcriptional activation in multiple colon cancer cells. ETAR stimulation acted via downstream G-protein Gαq/11 and Rho GTPase to suppress the Hippo pathway, thus leading to YAP/TAZ activation, which was required for ETAR-induced tumorigenesis. Overall, these results indicate a critical role of the YAP/TAZ axis in ETAR signaling. Cancer Res; 77(9); 2413-23. ©2017 AACR.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinogénesis/genética , Neoplasias del Colon/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Receptor de Endotelina A/genética , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias del Colon/patología , Endotelina-1/administración & dosificación , Endotelina-1/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rho/genéticaRESUMEN
Obstructive sleep apnea (OSA) results in cardiac dysfunction and vascular endothelium injury. Chronic intermittent hypoxia (CIH), the main characteristic of OSAS, is considered to be mainly responsible for cardiovascular system impairment. This study is aimed to evaluate the role of endothelin-1(ET-1) system in coronary injury and cardiac dysfunction in CIH rats. In our study, Sprague-Dawley rats were exposed to CIH (FiO2 9% for 1.5 min, repeated every 3 min for 8 h/d, 7 days/week for 3 weeks). After 3 weeks, the left ventricular developed pressure (LVDP) and coronary resistance (CR) were measured with the langendorff mode in isolated hearts. Meanwhile, expressions of ET-1 and ET receptors were detected by immunohistochemical and western blot, histological changes were also observed to determine effects of CIH on coronary endothelial cells. Results suggested that decreased LVDP level combined with augmented coronary resistance was exist in CIH rats. CIH could induce endothelial injury and endothelium-dependent vasodilatation dysfunction in the coronary arteries. Furthermore, ET-1 and ETA receptor expressions in coronary vessels were increased after CIH exposure, whereas ETB receptors expression was decreased. Coronary contractile response to ET-1 in both normoxia and CIH rats was inhibited by ETA receptor antagonist BQ123. However, ETB receptor antagonist BQ788 enhanced ET-1-induced contractile in normoxia group, but had no significant effects on CIH group. These results indicate that CIH-induced cardiac dysfunction may be associated with coronary injury. ET-1 plays an important role in coronary pathogenesis of CIH through ETA receptor by mediating a potent vasoconstrictor response. Moreover, decreased ETB receptor expression that leads to endothelium-dependent vasodilatation decline, might be also participated in coronary and cardiac dysfunction.