RESUMEN
The subintimal infiltration with monocytes is crucially involved in the development of complex atherosclerotic plaques. Monocyte chemotactic protein-1 (MCP-1) and its receptor CCR2 are important for monocyte extravasation and formation of atherosclerotic lesions. However, mechanisms of monocyte persistence in atherosclerotic plaques remain to be elucidated. Flow cytometric analysis revealed that monocytoid Mono Mac 6 cells that had transmigrated endothelium towards a MCP-1 gradient expressed higher levels of CCR2 than the non-migratory fraction, while input cells were intermediate, suggesting that high CCR2 levels are essential for transendothelial chemotaxis. Pretreatment of Mono Mac 6 cells or isolated human blood monocytes with the inflammatory cytokine tumor necrosis factor (TNF)-alpha dose- and time-dependently reduced MCP-1-induced transendothelial chemotaxis, which was inhibited by the CCR2 receptor antagonist 9-76 analog. This was paralleled by a decrease in CCR2 surface protein and mRNA expression. as assessed by flow cytometry and reverse transcription-polymerase chain reaction, inferring that inhibition of monocyte transmigration was due to downregulation of CCR2 to levels insufficient for chemotaxis. In contrast, treatment of monocytes with oxidized low-density protein (oxLDL) containing oxidized lipids, such as cholesteryl linoleate 13-hydroxide. but not with LDL, increased CCR2 protein and mRNA expression. Notably, oxLDL counteracted the TNF-alpha-mediated downregulation of CCR2 and CCR2-dependent transendothelial chemotaxis. Macrophage-colony-stimulating factor hardly affected CCR2 expression and function, suggesting that differentiation was not responsible for effects on CCR2. In conclusion, TNF-alpha impairs MCP-1-induced transendothelial migration of monocytes by downregulating CCR2 which appears critical for migration. Exposure to oxLDL antagonized the effects of TNF-alpha, and may thus contribute to monocyte retention and perpetuation of a chronic inflammatory reaction in unstable atherosclerotic lesions.
Asunto(s)
Arteriosclerosis/fisiopatología , Quimiocina CCL2/fisiología , Endotelio Vascular/fisiopatología , Lipoproteínas LDL/farmacología , Monocitos/fisiología , Receptores de Quimiocina/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Movimiento Celular , Células Cultivadas , Quimiotaxis de Leucocito , Regulación hacia Abajo , Citometría de Flujo , Humanos , Oxidación-Reducción , Reacción en Cadena de la Polimerasa , Receptores CCR2RESUMEN
The effects of stearic acid (C18:0) and trans-fatty acids (trans-FAs) on measures of platelet function and prostacyclin (PGI2) production are poorly understood in humans. In this controlled dietary study, platelet function and endothelial PGI2 production were studied in healthy humans after they consumed diets rich in C18:0 or trans-FAs. For 5 weeks, 80 subjects consumed a baseline diet high in saturated FAs and were then switched to a diet containing 9.3% of energy as stearic acid or a diet containing 8.7 energy% as trans-FAs from hydrogenated vegetable oils for another 5 weeks. All diets contained 32.2 to 33.9 energy% fat, 14.6 to 15.8 energy% saturated plus trans-FAs, 12.2 to 12.5 energy% cis-monounsaturated, and 2.9 to 3.5 energy% polyunsaturated FAs. No significant differences between the C18:0 and trans-FA diets were found in the urinary excretion of 2,3-dinor-thromboxane B2 or 2,3-dinor-6-keto-prostaglandin F1alpha. In vitro production of thromboxane B2 by platelets as well as urinary excretion of beta-thromboglobulin were also similar after both diets. Collagen-induced in vitro aggregation was significantly enhanced after the C18:0 diet compared with the trans-FA diet (P=.02), whereas no differences between the diets were found with ADP. The results indicate similar effects of C18:0 and trans-FA diets on platelet activation and endothelial PGI2 production.
Asunto(s)
Plaquetas/efectos de los fármacos , Endotelio Vascular/metabolismo , Epoprostenol/biosíntesis , Ácidos Grasos/administración & dosificación , Ácidos Esteáricos/administración & dosificación , Adulto , Plaquetas/metabolismo , Plaquetas/fisiología , Dieta , Ácidos Grasos/sangre , Ácidos Grasos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cooperación del Paciente , Ácidos Esteáricos/farmacología , EstereoisomerismoRESUMEN
The current analytical methods for the various prostanoids require a separate and extended sample workup, derivatization, and gas chromatographic/mass spectrometric detection of each compound. Therefore, we developed and validated a rapid method for the common purification, derivatization, and GC/MS determination of 11-dehydrothromboxane B2, 2,3-dinor-6-keto-PGF1a, PGF2A, PGE2, PGD2, and isoprostanes in urine. A single reversed-phase solid-phase extraction step and modified reaction conditions yielded excellent sample purification at high recoveries and efficient derivatization for all compounds in one vial. The method allows, for the first time, the simultaneous quantification of these index metabolites of systemic thromboxane and prostacyclin synthesis, renal prostaglandin formation, and nonenzymatic in vivo lipid peroxidation in a single GC/MS run with high sensitivity and precision.