Nox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products

Vascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cell (VSMC) ROS production are unclear. We studied the source and downstream targets of AGE-mediated ROS and reactive nitrogen species production in these cells. Significant increases in superoxide production in AGE-treated VSMC were measured using lucigenin (7650 +/- 433 vs 4485 +/- 424 LU/10(6) cells, p < 0.001) or coelenterazine (277,907 71,295 vs 120,456 4140 LU/10(6) cells,p < 0.05) and confirmed by ESR spectroscopy. These signals were blocked by the flavin-containing oxidase inhibitor diphenylene iodonium (DPI). AGE-stimulated NF-kappa B activity was abolished by DPI and the superoxide scavenger MnTBAP. AGE.differentially regulated VSMC NADPH oxidase catalytic subunits, stimulating the transcription of Nox1 (201 +/- 12.7%, p < 0.0001), while having no effect on Nox4. AGE also increased 3-nitrotyrosine formation, which was inhibited by MnTBAP, DPI, or the NOS inhibitor L-NAME. Regarding the source of NO, AGE stimulated inducible nitric oxide synthase mRNA (1 vs 9.7 +/- 3.0,p=0.046), which was abolished by a NF-kappa B inhibitor, SOD, catalase, or siRNA against Noxl. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-kappa B-dependent mechanism involving ROS generation by a Noxl-based oxidase. (c) 2007 Elsevier Inc. All rights reserved.