| Obesity is associated with chronic inflammation and increased risk of developing cardiovascular disease (CVD). Obesity is also associated with nitric oxide (NO)-mediated vascular endothelial dysfunction (VED), an independent predictor of increased CVD risk in humans. Pro-inflammatory cytokines secreted by the adipose tissue, such as TNF-alpha, may contribute to VED through promotion of insulin resistance or directly through a reduction in endothelial NO synthase (eNOS) expression and/or phosphorylation. Sodium salicylate (Na-Sal) is a non-acetylated aspirin that inhibits the pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB) and activates the cellular metabolism regulator AMP-activated protein kinase (AMPK). AMPK is a known activator of eNOS. We tested the hypothesis that Na-Sal increases eNOS expression/phosphorylation in TNFalpha-stimulated endothelial cells through an AMPK-dependent mechanism. Human aortic endothelial cells (HAECs) incubated in vitro with TNF-alpha (10 ng/ml, 2 hrs) demonstrated decreased (vs. control) expression (via Western blotting) of eNOSser1177 phosphorylation (n=8; P<.01) and AMPKThr172 phosphorylation (n=8, P<0.05), indicating an impaired phenotype. Pre-treatment of HAECs with Na-Sal (5 mM, 30 min) prevented the TNFalpha-stimulated decrease in eNOSser1177 phosphorylation (vs. control, n=7; P=0.14) and AMPKThr172 phosphorylation (vs. control, n=9; P=0.42). The AMPK activator AICAR prevented eNOSser1177 phosphorylation down-regulation by TNF-alpha in a manner similar to Na-Sal (n=2, P=0.839). Co-treatment with the AMPK inhibitor compound C (10 &mgr;M, 30 min) abolished the ability of Na-Sal to prevent down-regulation of eNOSser1177 phosphorylation by TNF-alpha (vs. control, n=3; P<0.001). These data suggest that Na-Sal reverses the inflammation-mediated reduction of phosphorylated eNOSser1177 in endothelial cells in part through AMPK. |
