RESUMEN
Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.
Asunto(s)
Modelos Animales de Enfermedad , Molécula 1 de Adhesión Intercelular/metabolismo , Malformaciones Arteriovenosas Intracraneales/metabolismo , Malformaciones Arteriovenosas Intracraneales/cirugía , Radiocirugia/métodos , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Masculino , Ratones , Dosificación Radioterapéutica , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECT: Radiosurgical treatment of brain arteriovenous malformations (AVMs) has the significant shortcomings of being limited to lesions smaller than 3 cm in diameter and of a latency-to-cure time of up to 3 years. A possible method of overcoming these limitations is stimulation of thrombosis by using vascular targeting. Using an animal model of AVM, the authors examined the durability of the thrombosis induced by the vascular-targeting agents lipopolysaccharide and soluble tissue factor conjugate (LPS/sTF). METHODS: Stereotactic radiosurgery or sham radiation was administered to 32 male Sprague-Dawley rats serving as an animal model of AVM; 24 hours after this intervention, the rats received an intravenous injection of LPS/sTF or normal saline. The animals were killed at 1, 7, 30, or 90 days after treatment. Immediately beforehand, angiography was performed, and model AVM tissue was harvested for histological analysis to assess rates of vessel thrombosis. RESULTS: Among rats that received radiosurgery and LPS/sTF, induced thrombosis occurred in 58% of small AVM vessels; among those that received radiosurgery and saline, thrombosis occurred in 12% of small AVM vessels (diameter < 200 µm); and among those that received LPS/sTF but no radiosurgery, thrombosis occurred at an intermediate rate of 43%. No systemic toxicity or intravascular thrombosis remote from the target region was detected in any of the animals. CONCLUSIONS: Vascular targeting can increase intravascular thrombosis after radiosurgery, and the vessel occlusion is durable. Further work is needed to refine this approach to AVM treatment, which shows promise as a way to overcome the limitations of radiosurgery.
Asunto(s)
Malformaciones Arteriovenosas Intracraneales/cirugía , Radiocirugia/métodos , Trombosis/cirugía , Animales , Angiografía Cerebral , Malformaciones Arteriovenosas Intracraneales/diagnóstico por imagen , Masculino , Modelos Animales , Ratas , Ratas Sprague-Dawley , Trombosis/diagnóstico por imagen , Resultado del TratamientoRESUMEN
Although most small arteriovenous malformations (AVM) are curable, over 90% of large lesions are untreatable with current surgery or radiosurgery. Endothelial cells (EC) are believed to be pivotal in the resulting vascular changes after AVM are irradiated, although their role is not fully understood. Elucidating the molecular effects of radiation on EC may allow development of new therapies that modulate the response of AVM to radiation. Cultured murine cerebral EC (bEnd.3) were exposed to a single 25 Gy dose of ionising radiation from a linear accelerator. Expression of the membrane proinflammatory and thrombotic molecules E-selectin, tissue factor (TF) and thrombomodulin (TM) were examined by immunofluorescent staining at times up to three weeks post irradiation. We found that E-selectin is significantly down regulated in the first 24 hours after irradiation. Later there is no significant difference in expression of this molecule between irradiated and non-irradiated groups. TM expression was significantly increased at all times, and the staining intensity of TF remained unchanged three weeks post irradiation. These results contribute to a greater understanding of the proinflammatory and thrombotic changes caused by irradiating normal brain EC.
Asunto(s)
Encéfalo/citología , Células Endoteliales/efectos de la radiación , Radiación , Radiocirugia/efectos adversos , Animales , Línea Celular Transformada , Supervivencia Celular , Relación Dosis-Respuesta en la Radiación , Selectina E/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Modelos Animales , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Factores de TiempoRESUMEN
Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 µM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes.
Asunto(s)
Antioxidantes/farmacología , Azoles/química , Azoles/farmacología , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Sincrotrones , Animales , Antioxidantes/química , Línea Celular , Isoindoles , Ratones , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Espectrometría por Rayos X , Células Tumorales CultivadasRESUMEN
Cultured neurons tolerate low H(2)O(2) concentrations (< or =50 microM) through the activity of constitutive antioxidant response elements (ARE). At H(2)O(2) levels (> or =100 microM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 microM H(2)O(2) enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H(2)O(2)-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-K(ATP) channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-K(ATP) channel (glybenclamide) inhibited the H(2)O(2)-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin's ability to bolster the intracellular pool of ATP in response to added H(2)O(2) is central to the preservation of cytoskeletal integrity and cell viability.
Asunto(s)
Globinas/metabolismo , Peróxido de Hidrógeno/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales KATP/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Globinas/genética , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Neuroglobina , Neuronas/citología , Neuronas/efectos de los fármacosRESUMEN
Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured human neuronal cells exposed to experimental hypoxia-re-oxygenation (H/R) injury responded with an increased production of reactive oxygen species (ROS) and a significant decrease in intracellular ATP. Expression of genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), glucose transporter-1 (Glut-1), the oxygen-sensor neuroglobin (Nb) and Cu,Zn-superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase-1 (Gpx-1) increased significantly in response to the insult. Enhanced expression of HO-1, SOD1 and CAT correlated with an increase in the corresponding protein activity. Despite the cellular response to bolster antioxidant capacity, apoptosis and necrosis increased following H/R injury. In contrast, ROS accumulation, the endogenous gene response and cell death was limited in neuronal cells pre-incubated with 50 or 100, but not 10 microM of the phenolic antioxidant 3,3',5,5'-tetra-t-butyl-biphenyl-4,4'-diol (BP) prior to H/R injury. These data indicate that the early endogenous gene response to H/R injury is unable to inhibit neuronal dysfunction and that increasing cellular antioxidant capacity with a synthetic polyphenol (>10 microM) is potentially neuro-protective.
Asunto(s)
Flavonoides/administración & dosificación , Oxigenoterapia Hiperbárica , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fenoles/administración & dosificación , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Anexinas/metabolismo , Caspasas/metabolismo , Catalasa/metabolismo , Diferenciación Celular , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flavonoides/química , Proteína GAP-43/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neuroblastoma/patología , Neuronas/metabolismo , Fenoles/química , Polifenoles , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Superóxidos/metabolismo , Factores de TiempoRESUMEN
Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia-reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin-adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), and the oxygen-sensor neuroglobin increased significantly. Up-regulation of the HO-1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti-oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti-oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti-oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro-protective drug.