Homocysteine induces VCAM-1 gene expression through NF-kB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants
Introduction: Hyperhomocysteinemia is a recognized risk factor for atherosclerotic vascular disease, but molecular mechanisms are no completely understood. Because VCAM-1 expression is crucial in monocyte adhesiveness, we evaluated the NF-κB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of specific dietetic polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT). Methods and Results: We found that in human umbilical vein endothelial cells (HUVEC), Hcy, at ≥50 μmol/L, but not cysteine, induced VCAM-1 expression at protein and mRNA levels, at enzyme immunoassay and Northern blot, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that two tandem NF-κB motives in VCAM-1 promoter are necessary for Hcy induced VCAM-1 gene expression. This was confirmed by Hcy induced NF-kB activation at EMSA and the nuclear translocation of its p65 (Rel A) subunit and the degradation of both inhibitors(I)kB-α and -b at Western analysis. Hcy increased intracellular ROS, measured with dichlorofluoresceine fluorescence assays, in HUVEC, by NAD(P)H oxidase activation, determined through the membrane translocation of its p47phox subunit. Antioxidants, NF-κB and NAD(P)H oxidase inhibitors decreased the Hcy inducted intracellular ROS and VCAM-1 expression. Previously, we found that antioxidants RSV and HT inhibited NF-κB mediated VCAM-1 induction by LPS or TNFα. Here we shown that nutritionally relevant concentrations of RSV and HT, but not folate and B vitamins, reduced (>60% inhibition at 10-6 mol/L) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. Conclusions: These data point out that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through NF-κB-mediated pro-oxidant mechanism. This can be inhibited by natural Mediterranean diet antioxidants, suggesting a their possible therapeutic role in Hcy-induced vascular damage
