Original Contribution
Mechanisms of H2O2-induced oxidative stress in endothelial cells

https://doi.org/10.1016/j.freeradbiomed.2006.02.017Get rights and content

Abstract

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H2O2 induces production of O2radical dot by activating NADPH oxidase. However, the mechanisms whereby H2O2 induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H2O2 on O2radical dot levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H2O2 markedly increased intracellular O2radical dot levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O2radical dot levels and attenuated cytotoxicity resulting from treatment with H2O2. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O2radical dot levels in PAEC treated with H2O2, suggesting that both NOS and NADPH oxidase contribute to H2O2-induced O2radical dot in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O2radical dot levels in PAEC treated with H2O2 to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H2O2 produces oxidative stress in endothelial cells by increasing intracellular O2radical dot levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H2O2 and oxidant-generating enzymes that may contribute to endothelial dysfunction.

Introduction

Oxidative stress causes endothelial dysfunction and cellular injury, which contribute to atherosclerosis [1] and other cardiovascular diseases [2]. O2radical dot is produced by a variety of cellular enzymes, including NADPH oxidase, xanthine oxidase, cyclooxygenase, cytochrome P450, and mitochondrial respiratory chain enzymes [2], [3], [4]. In addition, endothelial nitric oxide synthase (NOS) can produce large amounts of O2radical dot when the enzyme becomes uncoupled from its normal substrates [5], [6], [7], [8], [9]. O2radical dot is converted to H2O2 spontaneously or through the action of superoxide dismutase. O2radical dot, H2O2, and their reaction products modulate numerous aspects of vascular cell function.

H2O2 in the plasma is kept at low levels because of reactions with heme proteins, sulfhydryl groups, and ascorbate, suggesting that vascular endothelial cells encounter little circulating H2O2 [10]. However, H2O2 is a relatively stable ROS that is capable of diffusing through cellular membranes. Thus, it is likely that endothelial cells are exposed to substantially more H2O2 generated from intimal SMC and inflammatory cells in the subendothelial space. While the concentration of H2O2 in atherosclerotic blood vessels is not known, levels of H2O2 can exceed 100 μM in inflamed tissues [10]. This may be pertinent to sites of intense inflammation in atherosclerotic blood vessels.

The mechanisms by which H2O2 induces vascular cell injury are not fully understood. H2O2 does not contain an unpaired electron and is therefore less reactive than many other ROS. Thus, mechanisms other than direct oxidant injury likely contribute to the cytotoxic effects of H2O2 in vascular cells. In this regard, H2O2 reacts with peroxidases, such as myeloperoxidase, to form highly reactive molecules including HOCl [11] and nitrosylating species [12]. Additionally, there is increasing evidence that H2O2 can activate signaling pathways to stimulate ROS production in vascular cells. In SMC, H2O2 activates NADPH oxidase, resulting in the production of O2radical dot, and, consequently, oxidant injury [13].

However, it remains to be established whether this mechanism is also operative in endothelial cells. Moreover, in endothelial cells, H2O2 has been reported to stimulate NOS expression and activity [4]. It is plausible that H2O2-induced oxidative stress could lead to NOS uncoupling, which could in turn generate O2radical dot [14], [15], [16], although this has not been demonstrated experimentally.

Accordingly, the current report investigates the mechanisms of H2O2-induced oxidative stress in porcine aortic endothelial cells. Experiments were performed under static and shear conditions in order to gain insight into the potential modulatory influence of shear on the actions of H2O2 in endothelial cells.

Section snippets

Porcine aortic endothelial cell culture

Porcine aortic endothelial cells (PAEC) were obtained from the University of Iowa Cardiovascular Research Center Cell Culture Facility. They were cultured in Medium 199 (Invitrogen, M199) supplemented with 1% penicillin-streptomycin (Invitrogen) and 10% fetal bovine serum (Hyclone, FBS), with ascorbate levels in the physiologic range [17], [18]. Cultures were maintained at 37°C with 95% humidity and 5% CO2. Experiments were conducted in 24-well plates, at a density of 40,000 cells/well seeded

Porcine aortic endothelial cells produce O2 on exposure to H2O2

In SMC, H2O2 and lipid hydroperoxide species such as 13-HPODE have been demonstrated to increase O2radical dot levels, which in turn contributes to H2O2-induced cytotoxicity [13], [24], [25]. To determine whether H2O2 increases O2radical dot levels in endothelial cells, PAEC were treated with vehicle or 60 μmol/L H2O2 for 1.5 h, after which the presence of O2radical dot was examined using DHE. In vehicle-treated PAEC, only 4.9 ± 0.6% of nuclei exhibited DHE fluorescence. Following exposure of PAEC to H2O2, DHE fluorescence

Discussion

Three major conclusions can be drawn from this study: (1) In PAEC, H2O2 exposure increased intracellular O2radical dot, which caused cytotoxicity to the endothelial cells. (2) Both NOS and NADPH oxidase contributed to the increased O2radical dot levels induced by H2O2 in PAEC. (3) The combination of the NADPH oxidase inhibitor apocynin and L-sepiapterin abolishes H2O2-induced O2radical dot levels in PAEC.

The stimuli for ROS production in the vasculature are diverse and include cytokines and growth factors such as

Acknowledgments

This study was supported in part by NIH Grants HL-62984, HL-070860, HL-076684, and CA-086862; DOE DE-FG02-02ER63447; by a VA Merit review; and by the University of Iowa Biosciences Initiative Fund. The authors acknowledge Ms. Papri Chatterjee, Mr. John McRae, and Mr. Scott Mendralla for technical assistance.

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