RESUMEN
We have designed a multi-pinhole collimator for a dual-headed, stationary SPECT system that incorporates high-resolution silicon double-sided strip detectors. The compact camera design of our system enables imaging at source-collimator distances between 20 and 30 mm. Our analytical calculations show that using knife-edge pinholes with small-opening angles or cylindrically shaped pinholes in a focused, multi-pinhole configuration in combination with this camera geometry can generate narrow sensitivity profiles across the field of view that can be useful for imaging small objects at high sensitivity and resolution. The current prototype system uses two collimators each containing 127 cylindrically shaped pinholes that are focused toward a target volume. Our goal is imaging objects such as a mouse brain, which could find potential applications in molecular imaging.
Asunto(s)
Tomografía Computarizada de Emisión de Fotón Único/instrumentación , Animales , Fenómenos Biofísicos , Encéfalo/anatomía & histología , Encéfalo/diagnóstico por imagen , Diseño de Equipo , Imagenología Tridimensional , Ratones , Modelos Teóricos , Fantasmas de Imagen , Dispersión de Radiación , Tomografía Computarizada de Emisión de Fotón Único/métodos , Tomografía Computarizada de Emisión de Fotón Único/estadística & datos numéricosRESUMEN
Indirect evidence suggests accelerated degradation of endothelium-derived nitric oxide (ENDO) by superoxide anion (O2-) in hypercholesterolemic vessels (HV). To directly measure O2- production by normal vessels (NV) and HV, we used an assay for O2- based on the chemiluminescence (CL) of lucigenin (L). HV (1 mo cholesterol-fed rabbits) produced threefold more O2- than NV (1.47 +/- 0.20 nM/mg tissue/min, n = 7 vs. 0.52 +/- 0.05 nmol/mg tissue/min, n = 8, P < 0.001). Endothelial removal increased O2- production in NV (0.73 +/- 0.08, n = 6, P < 0.05), while decreasing it in HV (0.76 +/- 0.15, n = 5, P < 0.05). There was no difference between denuded HV and denuded NV. Oxypurinol, a noncompetitive inhibitor of xanthine oxidase, normalized O2- production in HV, but had no effect in NV. In separate isometric tension studies treatment with oxypurinol improved acetylcholine induced relaxations in HV, while having no effect on responses in normal vessels. Oxypurinol did not alter relaxations to nitroprusside. Thus, the endothelium is a source of O2- in hypercholesterolemia probably via xanthine oxidase activation. Increased endothelial O2- production in HV may inactivate endothelium-derived nitric oxide and provide a source for other oxygen radicals, contributing to the early atherosclerotic process.
Asunto(s)
Endotelio Vascular/metabolismo , Hipercolesterolemia/metabolismo , Superóxidos/metabolismo , Acridinas , Animales , Aorta/metabolismo , Colesterol/sangre , Colesterol en la Dieta , Mediciones Luminiscentes , Masculino , Oxipurinol/farmacología , Conejos , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/metabolismoRESUMEN
Erythropoietin is a 30.4 kDa protein that is produced and secreted from the kidney in response to anemia and hypobaric hypoxia. Binding of EPO to its receptor (EPO-R) on bone marrow-derived erythroid progenitor cells results in the stimulation of red blood cell production. Evidence is accumulating however, that the biological effects of recombinant EPO therapy extend beyond the stimulation of erythropoiesis. The discovery that the EPO-R is expressed on vascular endothelial cells suggests that the vasculature may be a biological target of EPO. Indeed, several studies have now demonstrated that the protective effect of EPO administration involves the activation of the protein kinase B/Akt pathway which can protect cells from apoptosis. Future work is likely to provide further insight into the mechanisms by which EPO protects vascular endothelial cells from injury and give us a better understanding of the pharmacological doses that are required to achieve this protection.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Eritropoyetina/farmacología , Animales , Apoptosis/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Transducción de SeñalRESUMEN
Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 microgram/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 micromol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.
Asunto(s)
Caveolinas , Colesterol/farmacología , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Especies Reactivas de Oxígeno/fisiología , Animales , Aorta/citología , Aorta/enzimología , Aorta/metabolismo , Aorta/ultraestructura , Bovinos , Caveolina 1 , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Proteínas de la Membrana/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III , Fracciones Subcelulares/metabolismoRESUMEN
Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation (P<0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-DeltaE1). Overexpression of TFPI decreased vascular TF activity compared with viral control (P<0.01). Overexpression of TFPI inhibited neointimal formation (P=0.038), resulting in enhanced luminal area (P=0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.
Asunto(s)
Arteriosclerosis/etiología , Trombosis de las Arterias Carótidas/etiología , Lipoproteínas/fisiología , Tromboplastina/fisiología , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/terapia , Trombosis de las Arterias Carótidas/metabolismo , Trombosis de las Arterias Carótidas/terapia , Terapia Genética , Lipoproteínas/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Recent evidence suggests that some benefit from the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors may occur independent of lipid lowering. We aimed to determine the effect of simvastatin on coronary endothelial function, endothelial NO synthase (eNOS) expression, and oxidative stress in experimental hypercholesterolemia (HC) in the absence of cholesterol lowering. Pigs were randomized to 3 experimental groups: normal diet (N group), high cholesterol diet (HC group), and HC diet with simvastatin (HC+S group) for 12 weeks. Low density lipoprotein cholesterol was similarly increased in the HC and HC+S groups compared with the N group. In vitro analysis of coronary large- and small-vessel endothelium-dependent vasorelaxation was performed. The mean vasorelaxation of epicardial vessels to bradykinin was significantly attenuated in the HC group compared with the N group (32.3+/-1.2% versus 42.9+/-1.6%, respectively; P<0.0001). This attenuation was significantly reversed in the HC+S group (38.7+/-1.5%, P<0.005 versus HC group). The maximal vasorelaxation to substance P was significantly attenuated in the HC group compared with the N group (50.5+/-11.9% versus 79.3+/-5.3%, respectively; P<0.05). This attenuated response was normalized in the HC+S group (74.9+/-4.1%, P<0.05 versus HC group). The maximal arteriolar vasorelaxation to bradykinin was also significantly attenuated in the HC group compared with the N group (71.9+/-4.9% versus 96.8+/-1.34%, respectively; P<0.005). This was reversed in the HC+S group (98.4+/-0.6%, P<0.0001 versus HC group). Western blotting of coronary tissue homogenates for eNOS demonstrated a decrease in protein levels in the HC group compared with the N group, with normalization in the HC+S group. Elevation of plasma F(2)-isoprostanes and thiobarbituric acid-reactive substances, markers of oxidative stress, occurred in the HC compared with the N group. These changes were reversed in the HC+S group. In summary, simvastatin preserves endothelial function in coronary epicardial vessels and arterioles in experimental HC (in the absence of cholesterol lowering) in association with an increase in coronary eNOS levels and a decrease in oxidative stress. These alterations may play a role in the reduction in cardiac events after treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiopatología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Hipercolesterolemia/prevención & control , Hipercolesterolemia/fisiopatología , Metabolismo de los Lípidos , Simvastatina/farmacología , Animales , Arteriolas/metabolismo , Arteriolas/fisiopatología , Western Blotting , Bradiquinina/metabolismo , Catalasa/análisis , Vasos Coronarios/enzimología , Dinoprost/análogos & derivados , Dinoprost/sangre , Endotelio Vascular/enzimología , F2-Isoprostanos , Femenino , Glutatión Peroxidasa/análisis , Hidroximetilglutaril-CoA Reductasas/sangre , Hipercolesterolemia/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Lípidos/sangre , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo III , Porcinos , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Vasodilatación/efectos de los fármacosRESUMEN
Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.
Asunto(s)
Calcio/fisiología , Endotelio Vascular/fisiología , Proteínas Quinasas/fisiología , Transducción de Señal/fisiología , Calmodulina/fisiología , Moléculas de Adhesión Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Endotelio Vascular/citología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Unión al GTP/fisiología , Expresión Génica , Humanos , Integrinas/fisiología , Transporte Iónico , Modelos Cardiovasculares , Óxido Nítrico Sintasa/fisiología , Concentración Osmolar , Paxillin , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/fisiología , Fosfoproteínas/fisiología , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptor de Insulina/fisiología , Reología , Estrés Mecánico , Estrés Fisiológico/fisiopatología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
The objective of this study was to test the hypothesis that serum biotin concentration and biotin balance (consumed - [urinary output + fecal output]) measured as total avidin-binding substances (biotin + biotin metabolites) are responsive to changes in the proportions of dietary alfalfa meal and concentrate fed to sheep. Eight sheep (initial BW = 40 kg) consumed a pelleted alfalfa meal-based diet that had 95:5, 48:52, 23:77, or 9:91% alfalfa meal:concentrate ratios (DM basis) in a replicated 4 x 4 Latin square design with 20-d periods (10 d of acclimation, 7 d of adaptation, and a 3-d collection period with jugular blood drawn on the last day). Replacing alfalfa meal with concentrate in the pelleted diets decreased dietary concentrations of biotin proportionally. As the percentage of alfalfa meal in the diet decreased, there was a linear decrease in daily DM intake (1,128 to 901 g of DMI/d; P < 0.01), with a linear (P < 0.01) and quadratic (P < 0.01) increase in the apparent total-tract DM digestibility of diets (51.0 to 80.0%). The biotin consumed decreased with alfalfa meal proportion in the diet (linear, P < 0.01). Both fecal biotin concentration (linear, P < 0.01) and fecal biotin output (quadratic, P < 0.05) increased, reaching peaks at 23% alfalfa meal. Fecal biotin output was not correlated with biotin intake, DMI, or intake of digestible DM. Mean urinary output, urinary biotin concentration, urinary biotin output, and serum biotin concentration were not affected by treatments. Means of biotin balance were negative and revealed the same trends among treatments as did fecal output. Biotin balance was a quadratic (P < 0.05) function of decreasing alfalfa meal in the diet, with more negative values at the alfalfa meal:concentrate ratio of 23:77. Results suggest that the greatest synthesis of biotin in the total digestive tract occurs with diets of either 52 or 77% concentrate for sheep; however, research addressing the significance of biotin metabolites on biotin balance and plasma biotin pool is needed.
Asunto(s)
Biotina/administración & dosificación , Biotina/sangre , Heces/química , Medicago sativa , Rumen/metabolismo , Ovinos/metabolismo , Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Biotina/metabolismo , Biotina/orina , Digestión , Relación Dosis-Respuesta a Droga , Fermentación , Masculino , Medicago sativa/química , Distribución AleatoriaRESUMEN
We conducted simulations to compare the potential imaging performance for breast cancer detection with High-Purity Germanium (HPGe) and Cadmium Zinc Telluride (CZT) systems with 1% and 3.8% energy resolution at 140 keV, respectively. Using the Monte Carlo N-Particle (MCNP5) simulation package, we modelled both 5 mm-thick CZT and 10 mm-thick HPGe detectors with the same parallel-hole collimator for the imaging of a breast/torso phantom. Simulated energy spectra were generated, and planar images were created for various energy windows around the 140 keV photopeak. Relative sensitivity and scatter and the torso fractions were calculated along with tumour contrast and signal-to-noise ratios (SNR). Simulations showed that utilizing a ±1.25% energy window with an HPGe system better suppressed torso background and small-angle scattered photons than a comparable CZT system using a -5%/+10% energy window. Both systems provided statistically similar contrast and SNR, with HPGe providing higher relative sensitivity. Lowering the counts of HPGe images to match CZT count density still yielded equivalent contrast between HPGe and CZT. Thus, an HPGe system may provide equivalent breast imaging capability at lower injected radioactivity levels when acquiring for equal imaging time.
Asunto(s)
Mama/diagnóstico por imagen , Cadmio/química , Germanio/química , Procesamiento de Imagen Asistido por Computador/métodos , Fantasmas de Imagen , Cintigrafía/instrumentación , Telurio/química , Zinc/química , Simulación por Computador , Femenino , Humanos , Método de Montecarlo , Fotones , Tomografía de Emisión de Positrones/instrumentación , Tomografía de Emisión de Positrones/métodos , Relación Señal-RuidoRESUMEN
We have performed a theoretical study to explore the potential and limitations of synthetic collimation for SPECT imaging with stacked-detector acquisition (dual magnification). This study will be used to optimize SiliSPECT, a small-animal SPECT for imaging small volumes such as a mouse brain at high sensitivity and resolution. The synthetic collimation enables image reconstruction with a limited number of camera views and in the presence of significant multiplexing. We also developed a new formulation to quantify the multiplexed object sensitivity and investigated how this changes for different acquisition parameters such as number of pinholes and combinations of front and back detector distances for imaging objects as small as the mouse brain. In our theoretical studies, we were not only able to demonstrate better reconstruction results by incorporating two detector magnifications in comparison to either one alone, but also observed an improved image reconstruction by optimizing the detector-collimator distances to change the multiplexing ratio between the front and back detectors.
Asunto(s)
Encéfalo/citología , Aumento de la Imagen/instrumentación , Procesamiento de Imagen Asistido por Computador/instrumentación , Tomografía Computarizada de Emisión de Fotón Único/instrumentación , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Diseño de Equipo , Aumento de la Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
PURPOSE: P-selectin expression is significantly increased in tumor microvasculature following exposure to ionizing radiation. The purpose of this study was to image radiation-induced P-selectin expression in vivo using optical imaging and gamma camera imaging in a heterotopic lung cancer model by using ScFv antibodies to P-selectin. PROCEDURES: In vitro studies using endothelial cells were done using 3 Gy radiation and selected ScFv antibodies to P-selectin. In vivo studies were performed using Lewis lung carcinoma cells subcutaneously injected into the hind limbs of nude mice. Mice were treated with 6 Gy radiation and sham radiation 10 days post-inoculation. P-selectin expression was assessed with near-infrared imaging using Cy7 labeled antibody, and gamma camera imaging using( 111)In-DTPA labeled antibody. RESULTS: In vitro studies showed antibody binding to P-selectin in radiation treated endothelial cells. In vivo optical imaging and gamma camera imaging studies showed significant tumor-specific binding to P-selectin in irradiated tumors compared to unirradiated tumors. CONCLUSIONS: Optical imaging and gamma camera imaging are effective methods for visualizing in vivo targeting of radiation-induced P-selectin in lung tumors. This study suggests that fluorescent-labeled and radiolabeled ScFv antibodies can be used to target radiation-induced P-selectin for the tumor-specific delivery of therapeutic drugs and radionuclides in vivo.
Asunto(s)
Anticuerpos Antineoplásicos/metabolismo , Neoplasias Pulmonares , Selectina-P/metabolismo , Radioterapia/métodos , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta en la Radiación , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Masculino , Ratones , Ratones Desnudos , Dosis de Radiación , CintigrafíaRESUMEN
We investigated a scheme for concurrently detecting low- and high-energy emissions from (123)I with a stacked silicon double-sided strip detector (DSSD) and modular scintillation camera (Modcam) from the FastSPECT II design. We sequentially acquired both low- and high-energy emission images of an (123)I object with a prototype DSSD and a Modcam. A sandwich aperture increases spatial resolution in the low-magnification DSSD image via a smaller pinhole diameter and allows a higher magnification image on the Modcam. Molybdenum, the insert material, efficiently stops 20-30 keV photons due to its â¼20 keV K-edge. Theoretically, less than 10% of 159 keV photons interact in 0.035 cm thick sheet of molybdenum, while this thickness stops virtually all â¼30 keV photons. Thus, photons from both energy regions will be incident upon their respective detectors with little cross talk. With a multi-pinhole collimator, we can decode multiplexed images on the Modcam by making use of the lower-magnification DSSD image. This approach can provide an increase in system sensitivity compared to single-detector configurations. Using MCNP5 we examined the potential benefits and drawbacks of stacked detectors and the sandwich aperture for small-animal pinhole SPECT via the synthetic-collimator method. Simulation results encourage us to construct the novel aperture and use it with our new DSSDs designed for mounting in a transmission configuration.
RESUMEN
A procedure for the quantitation of l-epinephrine and the determination of the d-/l-epinephrine ratio in a pharmaceutical formulation containing l-epinephrine is described. The optical isomers of epinephrine were resolved by capillary electrophoresis with a buffer containing heptakis-(2,6-di-o-methyl)-beta-cyclodextrin. Quantitation was achieved with the use of an internal standard, 1-pseudoephedrine. Results with and without internal standard correction illustrate the improved reproducibility possible with an internal standard.
Asunto(s)
Epinefrina/análisis , Electroforesis/métodos , Soluciones Oftálmicas , Reproducibilidad de los Resultados , EstereoisomerismoRESUMEN
Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca(2+)-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Endotelio Vascular/enzimología , Estrés Mecánico , Alcaloides/farmacología , Animales , Western Blotting , Calcio/metabolismo , Bovinos , Células Cultivadas , Interpretación Estadística de Datos , Regulación hacia Abajo , Endotelio Vascular/citología , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Isoenzimas/metabolismo , Forbol 12,13-Dibutirato/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transducción de Señal , EstaurosporinaRESUMEN
Endothelium-derived relaxing factor (EDRF) is rapidly inactivated by radicals. Endothelial cells possess several antioxidant defense mechanisms. It is not clear which intrinsic antioxidant defense systems are important to preserve the release of biologically active EDRF. We impaired antioxidant defense in normal vascular tissue by inhibiting catalase activity with 3-amino-1,2,4-triazole (AT), superoxide dismutase with diethyldithiocarbamate (DETC), and by reducing glutathione content via inhibiting glutathione synthesis with L-buthionine-(S,R)-sulfoximine (BSO). Pretreatment of rabbit aorta in vitro with DETC markedly reduced endothelium-dependent relaxation in response to acetylcholine and calcium ionophore A23187 and, to a lesser extent, reduced endothelium-independent relaxation in response to nitroprusside. Pretreatment of cultured bovine aortic endothelial cells (BAEC) with DETC did not alter release of nitrogen oxides (measured by chemiluminescence), but, the effluent of pretreated cells showed marked depression in vasodilator activity (measured by bioassay). Pretreatment of rabbit aorta in vitro with AT did not alter endothelium-dependent and -independent relaxations. Pretreatment of BAEC with BSO did not alter the release of nitrogen oxides or the vasodilator activity. These results suggest that endothelial superoxide dismutase activity, but not catalase or glutathione, is necessary for the release of biologically active EDRF. An imbalance of the intrinsic superoxide dismutase and the production of superoxide anions may therefore predispose to impaired endothelium-dependent relaxations and alter vascular reactivity.
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Vasos Coronarios/fisiología , Endotelio Vascular/fisiología , Músculo Liso Vascular/fisiología , Óxido Nítrico/metabolismo , Superóxido Dismutasa/metabolismo , Acetilcolina/farmacología , Amitrol (Herbicida)/farmacología , Animales , Bradiquinina/farmacología , Butionina Sulfoximina , Calcimicina/farmacología , Catalasa/antagonistas & inhibidores , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Ditiocarba/farmacología , Endotelio Vascular/efectos de los fármacos , Femenino , Glutatión/antagonistas & inhibidores , Técnicas In Vitro , Masculino , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Conejos , Superóxido Dismutasa/antagonistas & inhibidores , Porcinos , Vasodilatación/efectos de los fármacosRESUMEN
We tested the hypothesis that lysophosphatidylcholine (lyso-PC) could activate protein kinase C in intact vascular segments and sought to examine some of the physiological consequences of this activation. In segments of rabbit aorta, the patterns of protein phosphorylation determined by two-dimensional electrophoresis stimulated by lyso-PC and 12-O-tetradecanoylphorbol 13-acetate (TPA) were similar. Activation of protein kinase C can stimulate superoxide anion (O2-) production in other tissues, and we found that lyso-PC-treated rabbit aortas produced twofold more O2- than control vessels. Calphostin C, a potent and specific inhibitor of protein kinase C, attenuated O2- production in lyso-PC-treated vessels but had no effect in control vessels. The effect of lyso-PC on O2- production was mimicked by TPA. In separate bioassay studies, release of the endothelium-derived vascular relaxing factor (EDRF) quantified by the response of detector vessels was markedly impaired after exposure of donor rabbit aortic segments to lyso-PC. After incubation with calphostin C, EDRF release in response to acetylcholine from lyso-PC-treated donor vessels was restored significantly. Thus, lyso-PC can activate protein kinase C in intact vessels, leading to an increase in O2- production. Activation of protein kinase C by lyso-PC may also play a role in altering the release of EDRF in response to acetylcholine. Increased O2- production in response to lyso-PC may have important consequences in the atherogenic process.
Asunto(s)
Endotelio Vascular/metabolismo , Lisofosfatidilcolinas/farmacología , Naftalenos , Proteína Quinasa C/metabolismo , Superóxidos/metabolismo , Animales , Aorta/metabolismo , Activación Enzimática , Compuestos Policíclicos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , ConejosRESUMEN
Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of beta 1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 micrograms/mL) and flow (shear stress, 12 dynes/cm2) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the beta 1 affinity-sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a beta 1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that "costimulatory" events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Integrina beta1/fisiología , Proteínas Tirosina Quinasas/fisiología , Anticuerpos Monoclonales , Células Cultivadas , Endotelio Vascular/citología , Humanos , Fosfoproteínas/biosíntesis , Flujo Sanguíneo Regional , Estrés Mecánico , Tirosina/metabolismoRESUMEN
Nitroglycerin is a commonly employed pharmacological agent which produces vasodilatation by release of nitric oxide (NO.). The mechanism by which nitroglycerin releases NO. remains undefined. Recently, glutathione S-transferases have been implicated as important contributors to this process. They are known to release NO2- from nitroglycerin, but have not been shown to release NO.. The present studies were designed to examine the role of endogenous glutathione S-transferases in this metabolic process. Homogenates of dog carotid artery were incubated anaerobically with nitroglycerin, and NO. and NO2- production was determined by chemiluminescence. The role of glutathione S-transferases was studied by incubating homogenates with nitroglycerin in the presence of 1 mM GSH or 1 mM S-hexyl-glutathione, a potent inhibitor of glutathione S-transferases. Homogenates released 163 pmol of NO./h per mg of protein from nitroglycerin, and 2370 pmol of NO2-/h per mg. Adding GSH decreased NO. production by 82% and increased NO2- production by 98%. S-Hexylglutathione inhibited glutathione S-transferase activity by 96% and decreased NO2- production by 78%, but had no effect on NO. release. A linear relationship between glutathione S-transferase activity and NO2- production was observed, whereas glutathione S-transferase activity and NO. release were unrelated. Western-blot analysis demonstrated that dog carotid vascular smooth muscle contained Pi and Mu forms of glutathione S-transferases, with a predominance of the former. Purified preparations of human Pi and rat Mu isoforms metabolized nitroglycerin only to NO2- and not to NO.. On the basis of these findings, we conclude that (1) glutathione S-transferases do not contribute to the bioconversion of nitroglycerin to NO., but instead act as a degradative pathway for nitroglycerin, and (2) the release of NO. from nitroglycerin is not dependent on the formation of NO2-.
Asunto(s)
Arterias Carótidas/metabolismo , Glutatión Transferasa/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Nitroglicerina/metabolismo , Animales , Arterias Carótidas/enzimología , Perros , Radicales Libres , Glutatión Transferasa/aislamiento & purificación , Técnicas In VitroRESUMEN
The endothelium-derived relaxing factor is rapidly inactivated by superoxide radicals, and atherosclerotic vessels generate excess radical species. We tested the hypothesis that an imbalance between intrinsic superoxide dismutase (SOD) activity and the generation of superoxide radicals in atherosclerotic arteries may result in augmented inactivation of endothelium-derived relaxing factor. Vascular SOD was increased in normal and cholesterol-fed (1% cholesterol for 4 months) rabbits approximately twofold by treatment with polyethylene-glycolated SOD (PEG-SOD; 41,000 units/kg/day i.m.) for 1 week. Aortic rings from these animals and nontreated control and atherosclerotic rabbits subsequently were studied in organ chambers. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187 were improved by PEG-SOD in atherosclerotic but not in normal rabbits. PEG-SOD pretreatment did not alter endothelium-independent relaxations to nitroprusside. Thus, treatment with PEG-SOD can partially restore impaired endothelium-dependent relaxation of atherosclerotic arteries. We conclude that generation of oxygen-derived radicals likely contributes to endothelial dysfunction of atherosclerotic arteries.
Asunto(s)
Colesterol/administración & dosificación , Endotelio Vascular/fisiología , Polietilenglicoles/farmacología , Superóxido Dismutasa/farmacología , Vasodilatación/efectos de los fármacos , Acetilcolina/farmacología , Animales , Vasos Sanguíneos/enzimología , Calcimicina/farmacología , Colesterol/farmacología , Dieta , Femenino , Masculino , Conejos , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: We have shown that hypercholesterolemia increases vascular superoxide anion (O2-) production, which could be responsible for augmented inactivation of endothelium-derived vascular relaxing factor. We sought to determine whether this increased vascular O2- production is due to infiltration of macrophages into the intima and whether dietary treatment of hypercholesterolemia normalizes O2- production. METHODS AND RESULTS: A specific and sensitive assay for O2- based on chemiluminescence of lucigenin was used; the amount of O2- produced by vascular ring segments was quantified based on known quantities of O2- produced by xanthine-xanthine oxidase standards. O2- production of aortic segments from normal rabbits (n = 9), cholesterol-fed rabbits (1% cholesterol diet for 1 month, n = 7), and rabbits fed a 1% cholesterol diet for 1 month followed by a normal diet for 1 month (regression rabbits, n = 5) was measured. At the end of these diets, serum cholesterol levels were 1.5 +/- 0.2, 26.0 +/- 3.9, and 1.8 +/- 0.5 mmol/L (58 +/- 6, 1000 +/- 150, and 71 +/- 19 mg/dL) in the normal, cholesterol-fed, and regression animals, respectively. Vessels from normal rabbits with endothelium produced 0.32 +/- 0.06 nmol O2-/mg dry wt per minute, whereas those without endothelium produced approximately twice as much O2- (0.66 +/- 0.12 nmol O2- mg dry wt per minute. Vessels with endothelium from cholesterol-fed rabbits produced 4.5-fold more O2- than vessels from normal animals. This increased production of O2- was normalized by endothelial removal. This increased production of O2- was not due to infiltration of macrophages in the intima, because there was no correlation between vascular O2- production and macrophage infiltration assessed by immunohistochemistry with use of a specific antibody against rabbit macrophage. O2- production by vessels from regression rabbits was similar to that observed in normal animals, and as in the normal rabbits, endothelial removal increased O2- production. Aortic rings from these animals also were studied in organ chambers. Dietary lowering of cholesterol dramatically improved vasodilator responses to acetylcholine and A23187 (P < .05 versus cholesterol-fed rabbits). CONCLUSIONS: Dietary lowering of cholesterol not only improves endothelium-dependent vascular relaxation but also normalizes endothelial O2- production. Decreases of O2- production by dietary lowering of cholesterol not only may improve vasomotor control but also may improve other aspects of vascular integrity in atherosclerosis.