RESUMEN
AIMS: Moderate wine consumption is associated with a significant reduction of cardiovascular mortality. The molecular basis of this phenomenon remains unknown. Platelet-derived growth factor (PDGF) is an important contributor to atherogenesis. We investigated the effects of selected red and white wines on PDGF receptor (PDGFR) signalling in rat and human vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: All red wines concentration dependently inhibited the ligand-induced tyrosine phosphorylation of the PDGFR, downstream signalling events such as mitogen activated protein (MAP) kinase activation (Erk 1/2) and induction of immediate early genes (Egr-1, c-fos), and PDGF-induced cellular responses, whereas all white wines had no effect. At concentrations achieved after wine consumption in humans, all red wines completely abolished PDGF-dependent VSMC proliferation and migration. Red wines also inhibited PDGFR phosphorylation in vascular tissue, and in human coronary smooth muscle cells. Quantitative analyses of all tested wines and of samples collected at various time points (Days 0-16) of the 'mash fermentation', which is only performed for red wine, revealed that flavonoids of the catechin family, which potently inhibit PDGFR signalling, are extracted from grape seeds and skins during this process and therefore accumulate specifically in red wine. The accumulation of flavonoids correlated with the inhibitory potency of red wines on PDGFR signalling. Furthermore, this procedure could be imitated by incubation of wines with shredded grape seeds, and flavonoid-enriched white wine inhibited the PDGFR as potently as red wines. CONCLUSION: Only red wines abrogate a critical pathogenic mechanism during atherogenesis, PDGFR signalling, in VSMCs. This effect is mediated by non-alcoholic constituents, which accumulate during the mash fermentation. Our findings offer a molecular explanation for the vasoprotective effects particularly of red wine. Therefore, future epidemiological studies should consider differential protective effects of red and white wine in vivo.
Asunto(s)
Fermentación , Flavonoides/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Fenoles/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Vino , Animales , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Flavonoides/análisis , Humanos , Masculino , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fenoles/análisis , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Polifenoles , Ratas , Ratas Endogámicas WKY , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Tiempo , Vino/análisisRESUMEN
Growth factor-dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor-tyrosine-kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein-tyrosine-phosphatases (PTPs) are additional candidate regulators of these processes. Using laser-capture-microdissection and quantitative RT-polymerase chain reaction (qRT-PCR), we investigated the layer-specific expression of the four platelet-derived growth factor (PDGF) isoforms, the PDGF-alpha and beta receptors, and five PTPs implied in control of PDGF-receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF-beta receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression--particularly, DEP-1 levels--appeared to be the dominating factor determining receptor-phosphorylation and VSMC proliferation. In support of these findings, cultured DEP-1(-/-) cells displayed increased PDGF-dependent cell signaling. Hyperactivation of PDGF-induced signaling was also observed after siRNA-down-regulation of DEP-1 in VSMCs. The results indicate a previously unrecognized role of PDGF-receptor-targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK-dependent tissue remodeling.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas Tirosina Fosfatasas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Túnica Íntima/metabolismo , Animales , Traumatismos de las Arterias Carótidas/fisiopatología , Proliferación Celular , Quimiotaxis , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Músculo Liso Vascular/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Túnica Íntima/patologíaRESUMEN
OBJECTIVE: Growth factor- and reactive oxygen species (ROS)-induced activation of VSMCs is involved in vascular disease. This study investigates whether inhibitory oxidation of protein tyrosine phosphatases (PTPs) contributes to signaling in VSMCs in vitro and in vivo, and analyzes whether ROS- and growth factor-dependent vascular smooth muscle cell (VSMC) signaling is blunted by antioxidants that are able to activate oxidized PTPs. METHODS AND RESULTS: Signaling induced by H2O2 and platelet-derived growth factor (PDGF) was analyzed in VSMCs with or without the antioxidants N-acetyl-cysteine (NAC) and tempol. Effects of antioxidants on PDGF-stimulated chemotaxis and proliferation were determined. In vivo effects of antioxidants were analyzed in the rat carotid balloon-injury model, by analyzing neointima formation, cell proliferation, PDGF beta-receptor status, and PTP expression and activity. NAC treatment prevented H2O2-induced PTP inhibition, and reduced H2O2- and ligand-induced PDGF beta-receptor phosphorylation, PDGF-induced proliferation, and chemotaxis of VSMCs. Antioxidants inhibited neointima formation and reduced PDGF receptor phosphorylation in the neointima and also increased PTP activity. CONCLUSIONS: PTP-inhibition was identified as an intrinsic component of H2O2- and PDGF-induced signaling in cultured VSMCs. The reduction in PDGF beta-receptor phosphorylation in vivo, and the increase in PTP activity, by antioxidants indicate activation of oxidized PTPs as a previously unrecognized mechanism for the antirestenotic effects of antioxidants. The findings thus suggest, in general terms, reactivation of oxidized PTPs as a novel antirestenotic strategy.
Asunto(s)
Antioxidantes/farmacología , Reestenosis Coronaria/metabolismo , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/efectos de los fármacos , Acetilcisteína/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Reestenosis Coronaria/genética , Óxidos N-Cíclicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Peróxido de Hidrógeno/farmacología , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Tirosina Fosfatasas/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Marcadores de SpinRESUMEN
Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17beta-estradiol (E2) on beta-PDGF receptor (beta-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 microM-0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 +/- 15.8% and 79.4 +/- 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter beta-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the beta-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-gamma, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the beta-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.