Activation of NF-κB by Palmitate in Endothelial Cells A Key Role for NADPH Oxidase-Derived Superoxide in Response to TLR4 Activation

Objective-We investigated whether NADPH oxidase-dependent production of superoxide contributes to activation of NF-kappa B in endothelial cells by the saturated free fatty acid palmitate. Methods and Results-After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 mu mol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a >2-fold increase in superoxide levels, an effect associated with activation of NF-kappa B signaling as measured by phospho-I kappa B alpha, NF-kappa B activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of 3 different interventions-pretreatment with superoxide dismutase (SOD), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA-attenuated palmitate-mediated NF-kappa B signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitate-mediated superoxide production was not affected by overexpression of a phosphorylation mutant I kappa B alpha (NF-kappa B super repressor) that blocks cellular inflammation downstream of IKK beta/NF-kappa B. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4(-/-) mice, compared to low-fat fed controls. Conclusions-These results suggest that NADPH oxidase-dependent superoxide production links palmitate-stimulated TLR4 activation to NF-kappa B signaling in endothelial cells. (Arterioscler Thromb Vasc Biol. 2009;29:1370-1375.)