Original ContributionNox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products
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 O2− 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 O2− 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. O2− and O2−-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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