Homocysteine induces VCAM-1 gene expression through NF-κB and NAD(P)H oxidase activation - protective role of Mediterranean diet polyphenolic antioxidants Running head: Endothelium-activating effects of homocysteine

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-B-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-B-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 µmol/L, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, at enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-B motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-B activation was confirmed by EMSA, by the nuclear translocation of its p65 (Rel A) subunit, and the degradation of both inhibitors(I)B-and-at Western analysis. Hcy also increased intracellular ROS by NAD(P)H oxidase activation, assessed by the membrane translocation of its p47 phox subunit. NF-B inhibitors decreased Hcy-induced intracellular ROS and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10-6 mol/L) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a pro-oxidant mechanism involving NF-B. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage.