RESUMEN
The natural vitamin E analog α-tocopheryl phosphate (αTP) modulates atherosclerotic and inflammatory events more efficiently than the unphosphorylated α-tocopherol (αT). To investigate the molecular mechanisms involved, we have measured plasma levels of αTP and compared the cellular effects of αT and αTP in THP-1 monocytes. THP-1 cell proliferation is slightly increased by αT, whereas it is inhibited by αTP. CD36 surface expression is inhibited by αTP within hours without requiring transport of αTP into cells, suggesting that αTP may bind to CD36 and/or trigger its internalization. As assessed by gene expression microarrays, more genes are regulated by αTP than by αT. Among a set of confirmed genes, the expression of vascular endothelial growth factor is induced by αTP as a result of activating protein kinase B (PKB/Akt) and is associated with increased levels of reactive oxygen species (ROS). Increased Akt(Ser473) phosphorylation and induction of ROS by αTP occur in a wortmannin-sensitive manner, indicating the involvement of phosphatidylinositol kinases. The induction of Akt(Ser473) phosphorylation and ROS production by αTP can be attenuated by αT. It is concluded that αTP and αT influence cell proliferation, ROS production, and Akt(Ser473) phosphorylation in an antagonistic manner, most probably by modulating phosphatidylinositol kinases.
Asunto(s)
Antioxidantes/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Monocitos/efectos de los fármacos , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/farmacología , Animales , Antígenos CD36/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Gliburida/farmacología , Humanos , Monocitos/citología , Monocitos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Porcinos , Porcinos Enanos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , alfa-Tocoferol/sangreRESUMEN
1. Many studies have evaluated the effectiveness of alpha-tocopherol (vitamin E) in the development and progression of cardiovascular diseases, with conflicting results reported on the protective effect of this anti-oxidant. 2. The present study examined the effectiveness of a novel tocopheryl phosphate mixture (TPm) compared with that of alpha-tocopherol (TA) on key pro-inflammatory markers involved in atherogenesis, including interleukin (IL)-1beta, IL-6, IL-8, plasminogen activator inhibitor-1, tumour necrosis factor-alpha and C-reactive protein (CRP), as well as vascular function and lesion development in rabbits fed a 2% cholesterol diet. 3. Treatment with TPm, incorporated into the rabbit food at four doses ranging from 60 to 360 mg/kg chow, resulted in a significant reduction in plasma levels of all pro-inflammatory cytokines and biomarkers that appeared to be somewhat dose dependent. Conversely, treatment with TA, at a dose equivalent to the highest dose of TPm used, only decreased plasma levels of CRP, IL-6 and IL-8. Both TPm and TA treatment significantly improved vascular function to a similar extent, although TPm was more effective in reducing lesion development. 4. The reduction in these key pro-inflammatory markers appears to follow the improvement in the atherogenic state of the animals, indicating that the anti-inflammatory properties of TPm may be potentially beneficial in inflammatory disease states.
Asunto(s)
Antioxidantes/uso terapéutico , Aterosclerosis/prevención & control , Citocinas/sangre , Hipercolesterolemia/complicaciones , alfa-Tocoferol/análogos & derivados , Animales , Antioxidantes/administración & dosificación , Aorta/efectos de los fármacos , Aorta/patología , Aorta/fisiopatología , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Biomarcadores/sangre , Citocinas/inmunología , Dieta Aterogénica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipercolesterolemia/sangre , Hipercolesterolemia/inmunología , Hipercolesterolemia/patología , Técnicas In Vitro , Lípidos/sangre , Masculino , Conejos , Resultado del Tratamiento , Vasodilatación/efectos de los fármacos , alfa-Tocoferol/administración & dosificación , alfa-Tocoferol/uso terapéuticoRESUMEN
Phosphorylation of alpha-tocopherol produces an entity with enhanced antiatherogenic properties. Troglitazone, an alpha-tocopherol derivative of a 2,4-thiazolidinedione nucleus, is an antidiabetic agent that shows fatal idiosyncratic hepatotoxicity, a property not shared by later agents. We investigated the effects of phosphorylation of troglitazone (to yield "phosphoglitazone") on the biochemical pharmacologic properties of troglitazone. We investigated its ability to act as a PPARgamma agonist and to inhibit 2 atherogenic properties of vascular smooth muscle cells (vSMC)-proliferation and proteoglycan synthesis. PPARgamma activity was assessed in a transfection assay. Proliferation was assessed by [H]-thymidine incorporation and cell counting and proteoglycan synthesis by [S]-sulfate incorporation using human vSMCs stimulated with platelet-derived growth factor (PDGF; 50 ng/mL) and transforming growth factor (TGF)-beta (2 ng/mL). Phosphoglitazone was a full agonist for PPARgamma with a potency and efficacy similar to troglitazone. Phosphoglitazone also inhibited cell proliferation and proteoglycan synthesis with potency similar to troglitazone. We conclude that phosphorylation retains the pharmacologic activity of troglitazone while decreasing its lipophilicity and therefore potentially its toxicity. A phosphorylated derivative of a 2,4-thiazolidinedione warrants further investigation as a potential new therapeutic agent for the treatment of insulin resistance and Type 2 diabetes.
Asunto(s)
Cromanos/farmacología , Hipoglucemiantes/farmacología , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromanos/efectos adversos , Cromanos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/química , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , PPAR gamma/metabolismo , Fosforilación , Proteoglicanos/biosíntesis , Proteoglicanos/efectos de los fármacos , Ratas , Tiazolidinedionas/efectos adversos , Tiazolidinedionas/química , Transfección , TroglitazonaRESUMEN
The effect of tocopheryl phosphate on atherosclerosis progression has been studied in rabbits, fed with a 2% cholesterol diet and compared with an equivalent amount of alpha-tocopheryl acetate. The results show that the atherosclerotic-preventing effect of the phosphate derivative was more pronounced than that of the acetate derivative. alpha-Tocopheryl phosphate was also more potent in diminishing the expression of CD36 than the acetate derivative.
Asunto(s)
Anticolesterolemiantes/administración & dosificación , Aterosclerosis/tratamiento farmacológico , Dieta Aterogénica , alfa-Tocoferol/análogos & derivados , Animales , Aterosclerosis/inducido químicamente , Aterosclerosis/metabolismo , Antígenos CD36/biosíntesis , Colesterol/administración & dosificación , Colesterol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Conejos , Tocoferoles , alfa-Tocoferol/administración & dosificaciónRESUMEN
We have detected alpha-tocopheryl phosphate in biological tissues including liver and adipose tissue, as well as in a variety of foods, suggesting a ubiquitous presence in animal and plant tissue. Alpha-tocopheryl phosphate is a water-soluble molecule that is resistant to both acid and alkaline hydrolysis, making it undetectable using standard assays for vitamin E. A new method was therefore developed to allow the extraction of both alpha-tocopheryl phosphate and alpha-tocopherol from a single specimen. We used ESMS to detect endogenous alpha-tocopheryl phosphate in biological samples that also contained alpha-tocopherol. Due to the significance of these findings, further proof was required to unequivocally demonstrate the presence of endogenous alpha-tocopheryl phosphate in biological samples. Four independent methods of analysis were examined: HPLC, LCMS, LCMS/MS, and GCMS. Alpha-tocopherol phosphate was identified in all instances by comparison between standard alpha-tocopheryl phosphate and extracts of biological tissues. The results show that alpha-tocopheryl phosphate is a natural form of vitamin E. The discovery of endogenous alpha-tocopheryl phosphate has implications for the expanding knowledge of the roles of alpha-tocopherol in biological systems.
Asunto(s)
Vitamina E/aislamiento & purificación , alfa-Tocoferol/análogos & derivados , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida/métodos , Cromatografía de Gases y Espectrometría de Masas , Masculino , Espectrometría de Masas/métodos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de Electrospray , alfa-Tocoferol/aislamiento & purificación , alfa-Tocoferol/metabolismoRESUMEN
The finding that alpha-tocopheryl phosphate is present in cells in small amounts, that it can be synthesized and hydrolyzed supports the hypothesis that alpha-tocopheryl phosphate might be a signaling molecule. The possible pathways needed for the synthesis, hydrolysis and signaling are considered in this hypothesis as well the possible extension of this reaction to additional molecules such as tocopherols and tocotrienols. A possible mechanism of action of other tocopherol esters (succinate and maleate) is also hypothesized.
Asunto(s)
Fenómenos Fisiológicos Celulares , Modelos Biológicos , alfa-Tocoferol/análogos & derivados , Humanos , Hidrólisis , Fosforilación , Transducción de Señal/fisiología , alfa-Tocoferol/metabolismoRESUMEN
The effect of a mixture of alpha-tocopheryl phosphate and di-alpha-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukaemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which alpha-tocopherol was equally inhibitory. TPm led in non-stimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low density lipoprotein surface binding and oxLDL uptake. In non-stimulated THP-1 cells, alpha-tocopherol had only very weak effects on these events. Contrary to alpha-tocopherol, TPm was cytotoxic to THP-1 cells at high concentrations. Thus, TPm is able to inhibit the major aggravating elements involved in the progression of atherosclerosis. The higher potency of TPm may be due to a better uptake of the molecule and to its intracellular hydrolysis, providing more alpha-tocopherol to sensitive sites. Alternatively, a direct effect of the phosphate ester on specific cell targets may be considered.
Asunto(s)
Arteriosclerosis/metabolismo , Regulación hacia Abajo/efectos de los fármacos , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/farmacología , Animales , Aorta/citología , Apoptosis/efectos de los fármacos , Arteriosclerosis/patología , Antígenos CD36/biosíntesis , División Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Fragmentación del ADN , Humanos , Inflamación/metabolismo , Inflamación/patología , Lipoproteínas LDL/antagonistas & inhibidores , Lipoproteínas LDL/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Músculo Liso Vascular/citología , ARN Mensajero/genética , Ratas , Transcripción Genética/efectos de los fármacosRESUMEN
The effect of a mixture of alpha-tocopheryl phosphate plus di-alpha-tocopheryl phosphate (TPm) was studied in vitro on two cell lines, RASMC (from rat aortic smooth muscle) and human THP-1 monocytic leukemia cells. Inhibition of cell proliferation by TPm was shown in both lines and occurred with TPm at concentrations lower than those at which alpha-tocopherol was equally inhibitory. TPm led in nonstimulated THP-1 cells to inhibition of CD36 mRNA and protein expression, to inhibition of oxidized low-density lipoprotein surface binding and oxLDL uptake. In nonstimulated THP-1 cells, alpha-tocopherol had only very weak effects on these events.