Original ContributionEarly determinants of H2O2-induced endothelial dysfunction
Introduction
Reactive oxygen species (ROS) have been shown to contribute to cardiovascular disease, but some oxidative stress, such as that produced in moderate exercise, is associated with a decreased risk of cardiovascular disease [1], [2], [3]. One possible explanation for this apparent paradox is that small amounts or episodic exposures to ROS increase the oxidative buffering capacity of the body, while larger amounts or repeated exposures overpower these compensatory mechanisms. The determinants of when compensatory mechanisms fail are unclear, however.
H2O2, most commonly produced when O2− is dismutated by superoxide dismutase (SOD), is the most stable and long lasting of the ROS [4]. It is likely to play a critical role in maintaining the balance between O2− and NO, since it has been shown to act as a signaling molecule for both NADPH oxidases and endothelial nitric oxide synthase (eNOS). Presumably a compensatory response, exposure of endothelial cells to H2O2 causes upregulation of eNOS after several hours [5] and, on a shorter time scale (i.e., several minutes), activates eNOS through phosphorylation [6]. On the other hand, continued exposure to H2O2 for an intermediate time period (30 min) results in reduced NO bioavailability [7]. In addition, H2O2 activates the NADPH oxidases through phosphorylation of the NADPH oxidase subunit p47phox [4] to produce ROS, suggesting that continued or repeated ROS exposure may lead to reduced NO by oxidation of BH4, an essential eNOS cofactor, resulting in dysfunctional, uncoupled eNOS [8]. In these experiments, we tested the determinants of reduced NO production upon continued H2O2 exposures.
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Cell culture
Bovine aortic endothelial cells (BAECs; Cell Systems, Kirkland, WA) were cultured on 0.05% gelatin using M199 media (Cellgro, Herndon, VA) containing 10% fetal bovine serum (FBS, Invitrogen-Gibco, Carlsbad, CA) and 1% of each of the following: penicillin/streptomycin (Cellgro), minimal essential media (MEM) vitamins (Cellgro), L-glutamine (Invitrogen-Gibco), and MEM amino acids (Invitrogen-Gibco). Experiments were performed at 37°C on passage 4 or 5 cells that were 60–90% confluent. p47phox
Results
Previously, we have shown that exposure of endothelial cells to H2O2 results in a transient, immediate increase in eNOS-dependent NO production [6], [12]. Suggesting endothelial dysfunction, this presumably compensatory increase in NO production began to decrease over 30 min of continued exposure (Fig. 1). After H2O2 exposure 1, the reduction in NO production was associated with an increase in O2− production and a decrease in cellular BH4 (Figs. 1C and D). To quantify the degree of endothelial
Discussion
Many disease states are associated with decreased NO and increased ROS, yet exposure to moderate levels of oxidative stress induces increased NO production and production capacity [5], [6], presumably compensatory mechanisms. Therefore, it seems likely that some failure of these mechanisms is associated with the progression of disease. In this study, we explored early events leading to endothelial dysfunction during H2O2 exposure. We demonstrate that endothelial NO production is increased
Acknowledgments
This study was supported by National Institutes of Health (NIH) Grants HL64828 and HL73753 (S.C.D.), Department of Veterans Affairs Merit grant (S.C.D.), and an American Heart Association Established Investigator Award (S.C.D.). Dr. Widder was supported by the Deutsche Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/8-97).
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