Original ContributionMechanisms of H2O2-induced oxidative stress in endothelial cells
Introduction
Oxidative stress causes endothelial dysfunction and cellular injury, which contribute to atherosclerosis [1] and other cardiovascular diseases [2]. O2− 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 O2− when the enzyme becomes uncoupled from its normal substrates [5], [6], [7], [8], [9]. O2− is converted to H2O2 spontaneously or through the action of superoxide dismutase. O2−, 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 O2−, 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 O2− [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 O2− levels, which in turn contributes to H2O2-induced cytotoxicity [13], [24], [25]. To determine whether H2O2 increases O2− levels in endothelial cells, PAEC were treated with vehicle or 60 μmol/L H2O2 for 1.5 h, after which the presence of O2− 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 O2−, which caused cytotoxicity to the endothelial cells. (2) Both NOS and NADPH oxidase contributed to the increased O2− levels induced by H2O2 in PAEC. (3) The combination of the NADPH oxidase inhibitor apocynin and L-sepiapterin abolishes H2O2-induced O2− 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.
References (52)
- et al.
The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases
Trends Pharmacol. Sci.
(2003) - et al.
Reactivity of tetrahydrobiopterin bound to nitric-oxide synthase
J. Biol. Chem.
(1999) - et al.
Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function
Biochem. Biophys. Res. Commun.
(1999) - et al.
Hydrogen peroxide in the human body
FEBS Lett.
(2000) - et al.
Myeloperoxidase potentiates nitric oxide-mediated nitrosation
J. Biol. Chem.
(2005) - et al.
H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury
J. Biol. Chem.
(2001) - et al.
The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples
J. Biochem. Biophys. Methods
(2004) - et al.
Improvement in adenoviral gene transfer efficiency after preincubation at +37 degrees C in vitro and in vivo
Mol. Ther.: J. Am. Soc. Gene Ther.
(2002) - et al.
Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide
Free Radic. Biol. Med.
(2003) - et al.
Activation of NAD(P)H oxidase by lipid hydroperoxides: mechanism of oxidant-mediated smooth muscle cytotoxicity
Free Radic. Biol. Med.
(2003)
An assay for superoxide dismutase activity in mammalian tissue homogenates
Anal. Biochem.
Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry
Anal. Biochem.
Inhibition of GTP cyclohydrolase I by pterins
Biochim. Biophys. Acta
Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway
J. Biol. Chem.
Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase
J. Biol. Chem.
Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase
J. Biol. Chem.
NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II
J. Biol. Chem.
Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion
J. Biol. Chem.
Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells
Free Radic. Biol. Med.
Role of oxidative stress in atherosclerosis
Am. J. Cardiol.
Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress
Circ. Res.
Superoxide in the vascular system
J. Vasc. Res.
Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension
J. Clin. Invest.
The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study
Biochem. J.
Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite
J. Clin. Invest.
Leukocyte-derived myeloperoxidase amplifies high-glucose–induced endothelial dysfunction through interaction with high-glucose–stimulated, vascular non-leukocyte-derived reactive oxygen species
Diabetes
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