RESUMEN
BACKGROUND AND PURPOSE: Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy. EXPERIMENTAL APPROACH: Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](i) (Fura-2) were measured in aortic segments from C57Bl6 mice. KEY RESULTS: Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K(+) solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) influx diminished, while inhibition (nifedipine, 1-100 nM) augmented the relaxing capacity of NO. CONCLUSIONS AND IMPLICATIONS: In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.
Asunto(s)
Aorta Torácica/fisiología , Calcio/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Óxido Nítrico/fisiología , Vasodilatación , Acetilcolina/farmacología , Animales , Aorta Torácica/metabolismo , Canales de Calcio Tipo L/fisiología , GMP Cíclico/fisiología , Hidrazinas/farmacología , Técnicas In Vitro , Espacio Intracelular/metabolismo , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Contracción Muscular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroglicerina/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/fisiologíaRESUMEN
Recent investigations have highlighted that endogenous anti-inflammatory mediators and immune regulating mechanisms are important for the resolution of inflammatory processes. A disruption of these mechanisms can be causally related not only to the initiation of unnecessary inflammation, but also to the persistence of several chronic inflammatory diseases. In asthma, chronic Th-2 driven eosinophilic inflammation of the airways is one of the central abnormalities. To date, elucidating the role of the different pro-inflammatory mediators involved in orchestrating the inflammatory processes in asthma has been the subject of intense research in both humans and animal models. However, the counter-regulatory mechanisms that co-determine the outcome in the contest of resolution vs persistence of the eosinophilic airway inflammation remain poorly understood. These are currently being investigated in animal models of chronic asthma. Elucidating these mechanisms is of relevance, since it can give rise to a new therapeutic approach in the treatment of chronic airway inflammation in asthmatics. This novel concept of treatment involves the stimulation of endogenous anti-inflammatory pathways, rather than solely antagonising the various pro-inflammatory mediators. Here, we review and discuss the current knowledge about these endogenous anti-inflammatory mediators in clinical and experimental asthma.
Asunto(s)
Asma/fisiopatología , Inflamación/fisiopatología , Animales , Enfermedad Crónica , Eosinofilia , HumanosRESUMEN
A young woman has started cancer treatment because of a Hodgkin's lymphoma. After four months of chemotherapy, a PET scan showed an unexplained hotspot in the right lower abdomen. This was later explained by an unsuspected pregnancy. Our case emphasizes the importance of a pregnancy test in all women in the reproductive age before starting cancer treatment.
Asunto(s)
Fluorodesoxiglucosa F18/farmacocinética , Corazón/diagnóstico por imagen , Corazón/embriología , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/radioterapia , Miocardio/metabolismo , Complicaciones Neoplásicas del Embarazo , Adolescente , Femenino , Humanos , Tomografía de Emisión de Positrones/métodos , Embarazo , Resultado del Embarazo , Radiofármacos/farmacocinéticaRESUMEN
Non-specific anti-inflammatory medication is actually the treatment of choice for controlling the T-helper type 2 (Th-2) cell-driven airway inflammation in asthma. The induction of counterbalancing Th-1 cell clones, long considered a promising approach for immunotherapy, has failed to fulfil its promise because of potentially detrimental side-effects. This is therefore probably not a valid option for the treatment of asthma. With the increasing awareness that active immune mechanisms exist to control inflammatory responses, interest rises to investigate whether these can be exploited to control allergen-induced airway disease. The induction of antigen-specific T cells with suppressive characteristics (regulatory T cells) is therefore a potentially interesting approach. These regulatory T cells mediate tolerance in healthy, non-atopic individuals and have the potential of becoming an effective means of preventing allergen-induced airway inflammation and possibly of suppressing ongoing allergic immune responses. Here we review the available knowledge about allergen-induced suppressive immunity obtained from animal models taking into account the different developmental stages of allergic airway disease.
Asunto(s)
Traslado Adoptivo/métodos , Asma/terapia , Modelos Animales de Enfermedad , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Alérgenos/administración & dosificación , Animales , Asma/inmunología , Humanos , Tolerancia Inmunológica , RatonesAsunto(s)
Enfermedades del Recién Nacido/prevención & control , Metástasis de la Neoplasia/prevención & control , Complicaciones Neoplásicas del Embarazo/tratamiento farmacológico , Complicaciones Neoplásicas del Embarazo/prevención & control , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Femenino , Feto/efectos de los fármacos , Humanos , Recién Nacido , EmbarazoRESUMEN
The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.
Asunto(s)
Pared Celular/ultraestructura , Helechos/citología , Helechos/ultraestructura , Hojas de la Planta/citología , Hojas de la Planta/ultraestructura , Diferenciación Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Helechos/anatomía & histología , Helechos/efectos de los fármacos , Histocitoquímica , Microscopía Electrónica , Concentración Osmolar , Enfermedades de las Plantas , Hojas de la Planta/anatomía & histología , Hojas de la Planta/efectos de los fármacos , Cloruro de Sodio/farmacologíaRESUMEN
All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Imidazoles/farmacología , Neoplasias Mamarias Experimentales/prevención & control , Oxigenasas de Función Mixta/antagonistas & inhibidores , Tiazoles/farmacología , Tretinoina/metabolismo , Animales , Benzotiazoles , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , División Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Ácido Retinoico 4-Hidroxilasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The decision to revise a scar should be based on risk versus benefit. Scars over 2 cm in length or scars greater than 2 mm in width can usually be improved with scar revision. Although many techniques exist, scar irregularization has been used for scar camouflage for many years. Two techniques commonly employed for scar irregularization are W-plasty and geometric broken line closure. W-plasty provides a regularly irregular scar, and geometric broken line closure provides an irregularly irregular scar. Each method can offer excellent results when performed correctly and in the appropriate situations. The advantages, disadvantages, and design of each method are discussed.
Asunto(s)
Cicatriz/cirugía , Procedimientos de Cirugía Plástica/métodos , Cara/cirugía , Humanos , ReoperaciónRESUMEN
One of the most commonly used techniques in facial plastic surgery is the Z-plasty. Main reasons to perform these transposition flaps are to lengthen a pre-existing scar, to camouflage a scar, or to realign a scar. The classic 60 degrees Z-plasty allows a 75% increase in scar length and is the cornerstone against which all variations are compared. Understanding the classic Z-plasty permits the surgeon to expand his or her repertoire to include the numerous variations thereof. The double-opposing Z-plasty, unequal triangle Z-plasty, four-flap Z-plasty, compound Z-plasty, and planimetric Z-plasty are the most frequent variants of the basic Z-plasty. Each are presented with illustrations and clinical indications.
Asunto(s)
Cicatriz/cirugía , Cara/cirugía , Procedimientos de Cirugía Plástica/métodos , Colgajos Quirúrgicos , HumanosRESUMEN
Focal adhesion kinase (FAK) plays an important role in integrin-mediated signal transduction pathways and its C-terminal noncatalytic domain Fak-related non-kinase (FRNK), which is autonomously expressed, acts as an inhibitor of FAK. A model has been proposed where FAK and FRNK compete for an essential common binding protein. A FRNK variant in which the direct interaction with v-Crk-associated tyrosine kinase substrate (CAS) was disturbed by point mutations still functioned as an inhibitor of FAK, suggesting that FRNK is unlikely to inhibit FAK by sequestering CAS. Deletion variants of FRNK within the region N-terminal to the focal adhesion targeting (FAT) sequence were still able to inhibit FAK function, indicating that this region is dispensable for the inhibitory effect of FRNK. Overexpression of a green fluorescent protein (GFP) fusion protein containing the FAT sequence delayed cell spreading and reduced FAK tyrosine phosphorylation. This indicates that the FAT sequence is the major inhibitory moiety within FRNK.
Asunto(s)
Adhesiones Focales/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Adhesión Celular , Células Cultivadas , Embrión de Pollo , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Endopeptidasas/fisiología , Fragmentos de Péptidos/biosíntesis , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/farmacología , Línea Celular , Medios de Cultivo/química , Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/antagonistas & inhibidores , Isoformas de Proteínas/análisis , Solubilidad , TransfecciónRESUMEN
The participation of prostanoids, nitric oxide and non-prostanoid non-nitric oxide factors in endothelium-dependent relaxations was investigated in phenylephrine (PE)-constricted carotid and femoral arteries of C57BL6 mice. The carotid artery was more sensitive to acetylcholine as compared to the femoral artery, and cyclooxygenase inhibition did not influence the relaxation in either vessel. In the carotid artery, high doses of acetylcholine caused transient constrictions, which were abolished by indomethacin or piroxicam. In the carotid but not the femoral artery, N(omega)-nitro-L-arginine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced PE-induced contractions enormously, suggesting that endogenous nitric oxide production is much higher in the carotid artery. While in the carotid artery all relaxation was abolished by N(omega)-nitro-L-arginine or ODQ, a residual response (34+/-5% and 74+/-4%, respectively) but with a different shape, was maintained in the femoral artery. This N(omega)-nitro-L-arginine-resistant relaxation was abolished by the combination of apamin and charybdotoxin. In both arteries, ODQ abolished relaxation to S-nitroso-N-acetyl-D-penicillamine, while N(omega)-nitro-L-arginine enhanced the sensitivity to this donor of exogenous nitric oxide. In 30 mM KCl, the relaxation to acetylcholine was abolished by N(omega)-nitro-L-arginine or ODQ in either artery. In conclusion, in the carotid artery endothelium-dependent relaxation is mediated predominantly by nitric oxide acting via cyclic GMP-dependent pathways, while in the femoral artery part of the relaxation can be attributed to a non-prostanoid non-nitric oxide factor operating via apamin/charybdotoxin-sensitive potassium channels.
Asunto(s)
Arterias Carótidas/fisiología , Arteria Femoral/fisiología , Vasoconstricción/fisiología , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Arginina/farmacología , Arterias Carótidas/efectos de los fármacos , Femenino , Arteria Femoral/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Fenilefrina/farmacología , Prostaglandinas/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
All-trans-retinoic acid (RA) regulates epithelial differentiation and growth through activation of specific nuclear RA receptors (RARs). Because high-rate metabolism largely impairs the biological efficacy of RA, we have sought for compounds capable of inhibiting the metabolic breakdown of the retinoid. This study identifies R115866 as a novel inhibitor of the cytochrome P450 (CYP)-mediated metabolism of RA. In vitro, nanomolar concentrations of R115866 inhibited the conversion of RA by CYP26, a RA-inducible RA metabolizing enzyme. In vivo, oral administration of R115866 (2.5 mg/kg) to rats induced marked and transient increases of endogenous RA levels in plasma, skin, fat, kidney, and testis. Consistent with its ability to enhance endogenous RA content in tissues, R115866 was found to exert retinoidal activities. Like RA, the title compound: 1) inhibited vaginal keratinization in estrogen-stimulated rats; 2) induced epidermal hyperplasia in mouse ear skin; 3) transformed mouse tail epidermis from a para- to an orthokeratotic skin type; and 4) up-regulated the CYP26 mRNA expression in rat liver. Furthermore, we found that the keratinization-suppressive and CYP26-inducing activities of R115866 could be reversed by concomitant administration of the RAR antagonist, AGN193109. Our data characterize R115866 as a potent, orally active inhibitor of RA metabolism, capable of enhancing RA levels and displaying retinoidal actions. These activities are reversed by RAR antagonism, supporting the idea that the actions of R115866 result from increased availability of endogenous RA and improved RAR triggering.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Retinoides/metabolismo , Tiazoles/farmacología , Tretinoina/metabolismo , Triazoles/farmacología , Animales , Inhibidores de la Aromatasa , Benzotiazoles , Sistema Enzimático del Citocromo P-450/genética , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Femenino , Humanos , Hiperplasia/inducido químicamente , Queratosis/inducido químicamente , Masculino , Ratones , Ovariectomía , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Vagina/metabolismoRESUMEN
The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378-2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14alpha-methyl-ergosta-8,24(28)-diene-3beta,6alpha-dio l (3, 6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14alpha-methyl-ergosta-5,7,22,24(28)-tetraene-3betaol, and 14alpha-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Itraconazol/farmacología , Cetosteroides/metabolismo , Transactivadores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Microbiana , Ergosterol/biosíntesis , Pruebas de Sensibilidad Microbiana , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Reacción en Cadena de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulador Transcripcional ERGRESUMEN
Oxidized low density lipoprotein (oxLDL) is present in atherosclerotic lesions and has been implicated in the etiopathogenesis of atherosclerosis mainly based on in vitro studies. In view of the lack of data on the activity of oxLDL in vivo, we decided to study its effects in the rabbit by local application at the level of the vascular wall. Intimal thickening was evoked by the placement of a silicone collar around the carotid arteries during 2 weeks. The collar was connected to an osmotic minipump containing human oxLDL (7 micrograms h-1), LDL (7 micrograms h-1) or phosphate-buffered saline. Collar placement resulted in a thickening of the intima thereby increasing the thickness from 5 +/- 1 to 26 +/- 5 microns with the appearance of alpha-actine positive smooth muscle cells. Perivascular infusion of LDL or oxLDL significantly enhanced the intima, containing large amounts of T-lymphocytes, collagen and smooth muscle cells. The placement of the collar and the infusion of oxLDL during 14 days resulted in an increased sensitivity to serotonin and a decreased sensitivity to acetylcholine. The maximal relaxation to acetylcholine was reduced by 50% whereas the endothelium-independent relaxation to nitroglycerin were not affected. These results show for the first time that the local application of oxLDL in vivo promotes intimal thickening and impairs the endothelium-dependent relaxations thereby supporting the suggestion that oxLDL plays an important role in the morphological and functional changes present in atherosclerotic blood vessels.
Asunto(s)
Lipoproteínas LDL/metabolismo , Túnica Íntima/metabolismo , Sistema Vasomotor/fisiología , Animales , Humanos , Oxidación-Reducción , ConejosRESUMEN
OBJECTIVES: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. METHODS: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu(2+)-oxidized, 7 micron/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. RESULTS: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11 +/- 2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98 +/- 16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (-logEC50 oxLDL 6.95 +/- 0.11 vs. 7.52 +/- 0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. CONCLUSION: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Túnica Íntima/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Acetilcolina/farmacología , Animales , Calcimicina/farmacología , Cobre/farmacología , Relación Dosis-Respuesta a Droga , Ionóforos/farmacología , Peroxidación de Lípido , Lipoproteínas LDL/metabolismo , Masculino , Nitroglicerina/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Fenilefrina/farmacología , Conejos , S-Nitroso-N-Acetilpenicilamina , Túnica Íntima/patología , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 micrograms h-1, n = 16 arteries), oxLDL (Cu2+ oxidized, 7 micrograms h-1, n = 16), or phosphate-buffered saline (5 microL h-1, n = 16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of alpha-SMC actin-immunoreactive cells in the intima, thereby increasing the intimal thickness from 5 +/- 1 to 26 +/- 5 microns. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5 +/- 2% in the discrete collar-induced intima to approximately 20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.
Asunto(s)
Arterias Carótidas/efectos de los fármacos , Estenosis Carotídea/patología , Lipoproteínas LDL/toxicidad , Túnica Íntima/efectos de los fármacos , Animales , Arterias Carótidas/patología , Estenosis Carotídea/etiología , Colágeno/análisis , Constricción , Humanos , Hiperplasia , Bombas de Infusión Implantables , Peroxidación de Lípido , Lipoproteínas LDL/farmacología , Masculino , Músculo Liso Vascular/patología , Conejos , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Túnica Íntima/química , Túnica Íntima/patologíaRESUMEN
Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.
Asunto(s)
Neoplasias de la Mama/metabolismo , Queratolíticos/metabolismo , Retinoides/farmacología , Tretinoina/metabolismo , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Citosol/efectos de los fármacos , Citosol/metabolismo , Femenino , Humanos , Imidazoles/farmacología , Oxidación-Reducción , Reproducibilidad de los Resultados , Proteínas de Unión al Retinol/efectos de los fármacos , Proteínas de Unión al Retinol/metabolismo , Células Tumorales CultivadasRESUMEN
We studied the enzymatic characteristics of the oxidative catabolism of retinoic acid (RA) and its inhibition by liarozole-fumarate in homogenates of rat Dunning R3327G prostate tumors. Homogenates of rat liver were used as reference material. Both tumor and liver homogenates were able to catabolize retinoic acid. HPLC analysis revealed only very polar metabolites in tumors, while in the liver both metabolites with intermediate polarity and more polar metabolites were found. Kinetic analysis of retinoic acid catabolism showed a K(m) of 1.7 +/- 0.7 microM and a Vmax of 4.2 +/- 4.4 pmol polar RA metabolites/mg protein/hr for Dunning G tumor homogenates. In liver homogenates a K(m) value of 4.3 +/- 0.5 microM and a Vmax value of 290 +/- 120 pmol polar RA metabolites/mg protein/hr were obtained. Liarozole-fumarate inhibited retinoic acid catabolism in Dunning tumors and liver with IC50 values of 0.26 +/- 0.16 microM and 0.14 +/- 0.05, respectively. The results suggest that rat Dunning R3327G tumors are able to metabolize retinoic acid in a manner similar to that found in rat liver but with a lower metabolizing capacity.