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

https://doi.org/10.1016/j.freeradbiomed.2007.02.002Get rights and content

Abstract

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/106 cells, p < 0.001) or coelenterazine (277,907 ± 71,295 vs 120,456 ± 4140 LU/106 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-κB 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-κB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-κB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

Section snippets

Cell culture

VSMC were isolated from the thoracic aorta of male Sprague–Dawley rats by an enzymatic digestion protocol with modifications [29]. Cells between the third and the sixth passage were cultured in low-glucose DMEM (Sigma, St. Louis, MO, USA) supplemented with 10% calf serum. Quiescence was achieved by serum-starvation in 0.01% serum medium for 48 h before treatments.

Incubation with AGE

AGE were obtained by long-term exposure of albumin to glucose [30]. BSA at 50 mg/ml (low endotoxin, fatty acid free; Sigma) was

AGE induce superoxide production in VSMC

To evaluate the capacity of AGE to stimulate superoxide production at different concentrations, a dose-response curve for ROS generation was performed using various concentrations of AGE (1–500 μg/L). As is shown in Fig. 1, AGE are potent stimulators of O2radical dot production over a narrow dose range, with an EC50 of ∼ 30 μg/ml and a maximal response attained at 50–300 μg/ml. Concentrations below 20 μg/L failed to induce O2radical dot production. An AGE concentration of 200 μg/L was used for further experiments.

Discussion

Activation of VSMC, the most abundant cell type in the vessel wall, plays a central role in the progression of diabetic macroangiopathy. O2radical dot and O2radical dot-derived H2O2 have been shown to have profound effects on VSMC migration, growth, and differentiation [15], [41], [42], [43]. Enhanced intracellular oxidative stress after exposure to AGE has been reported [8], [9], [10]; however, the identity and primary sources of these ROS are not clear. Using different approaches, the present study shows that

Acknowledgments

This work was supported by Projects PUC-PBMEC 2001–2003, Fondecyt 1020486, HL075209 and FAPESP 00/12154-2. The authors thank Drs. Ohara Agusto, Sergei Dikalov, and Marcelo Pedro for their technical help with EPR measurements and Dr. Victoria Velarde for her valuable discussions during the performance of these studies.

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