RESUMEN
Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However, the underlying mechanism of endothelial cell injury in hyperhomocysteinemia has not been elucidated. In this study, we examined the effect of homocysteine (Hcy) on Fas-mediated apoptosis in endothelial cells. Hcy-induced upregulation of Fas in endothelial cells (ECs) in a dose-dependent manner. At the same time, Hcy increased intracellular peroxide in ECs. Hcy-induced Fas expression was inhibited by the treatment with catalase. Hcy increased NF-kappaB DNA binding activity, and adenovirus-mediated transfection of a Ikappa-B mutant (Ikappa-B mt) gene inhibited Hcy-induced Fas expression. ECs were sensitive to Fas-mediated apoptosis when exposed to Hcy. Under these condition, Ikappa-B mt protected ECs from Fas-mediated apoptosis. In addition, Hcy inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP). Adenovirus-mediated transfection of constitutively active Akt gene abolished the Hcy-mediated downregulation of FLIP. These data suggest that upregulation of Fas expression and downregulation of FLIP is a mechanism through which Hcy induces EC apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Homocisteína/toxicidad , Péptidos y Proteínas de Señalización Intracelular , Receptor fas/fisiología , Animales , Arteriosclerosis/etiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/fisiología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiología , Ratones , FN-kappa B/fisiología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Venas Umbilicales/citología , Regulación hacia Arriba/efectos de los fármacos , Receptor fas/biosíntesis , Receptor fas/genéticaRESUMEN
Dietary supplementation with eicosapentaenoic acid (EPA) improves the prognosis of chronic inflammatory diseases, including atherosclerosis. The mechanism underlying these beneficial effects, however, remains to be elucidated. Here we show that EPA protects endothelial cells from anoikis through upregulation of the cellular FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme)-inhibitory protein (cFLIP), an endogenous inhibitor of caspase-8. EPA-induced upregulation of cFLIP expression was partially suppressed by the phosphatidylinositol-3-kinase inhibitor wortmannin. Conversely, treatment with insulinlike growth factor-1 (IGF-1), an activator of phosphatidylinositol-3-kinase/Akt signaling, or infection with an adenoviral construct expressing the constitutively active Akt gene induced upregulation of cFLIP expression. In addition, pretreatment of endothelial cells with either EPA or IGF-1 protected them from anoikis, suggesting that EPA-induced protection against anoikis is partially mediated through activation of Akt. On the other hand, when endothelial cells were already detached, treatment of these cells with EPA but not with IGF-1 protected them against anoikis. Importantly, EPA restored cFLIP expression without activating Akt signaling in detached endothelial cells, whereas IGF-1 had no effect. Additionally, exogenously restored expression of cFLIP by the tetracycline-regulated adenovirus system protected endothelial cells against anoikis. Furthermore, EPA was protective against the loss of endothelium in an organ culture of rat aortas. These findings suggest that EPA protects against endothelial cell anoikis through restoration of cFLIP expression, which might contribute to the mechanism underlying the beneficial effects of EPA in patients with hypertension.