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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1370.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Activation of NF-B by Palmitate in Endothelial Cells

A Key Role for NADPH Oxidase-Derived Superoxide in Response to TLR4 Activation

Ezekiel Maloney; Ian R. Sweet; David M. Hockenbery; Matilda Pham; Norma O. Rizzo; Sanshiro Tateya; Priya Handa; Michael W. Schwartz; Francis Kim

From the Department of Medicine (E.M., I.R.S., D.M.H., M.P., N.O.R., S.T., P.H., M.W.S., F.K.), the Diabetes and Obesity Center of Excellence (I.R.S., N.O.R., S.T., P.H., M.W.S., F.K.), and the Fred Hutchinson Cancer Research Center (D.M.H.), University of Washington, Seattle.

Correspondence to Francis Kim, Department of Medicine, 815 Mercer St, Box 358055, University of Washington, Seattle, WA 98109. E-mail fkim{at}u.washington.edu

Objective— We investigated whether NADPH oxidase–dependent production of superoxide contributes to activation of NF-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 µmol/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-B signaling as measured by phospho-IB, NF-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-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 IB (NF-B super repressor) that blocks cellular inflammation downstream of IKKβ/NF-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-B signaling in endothelial cells.

These studies were undertaken to delineate the pathways linking TLR4 activation to the activation of IKKβ/NF-B signaling in vascular tissue. We demonstrate in cultured human endothelial cells that superoxide production is required for palmitate to induce NF-B signaling, and further that palmitate increases superoxide production by activating NADPH oxidase through the TLR4 signaling pathway.

Key Words: toll like receptor 4 • NADPH oxidase • endothelial cells • vascular inflammation