Elsevier

Free Radical Biology and Medicine

Volume 39, Issue 9, 1 November 2005, Pages 1238-1248
Free Radical Biology and Medicine

Original Contribution
Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures

https://doi.org/10.1016/j.freeradbiomed.2005.06.010Get rights and content

Abstract

Endothelial dysfunction/activation underlies the development of long-term cardiovascular complications and atherosclerosis. The aim of this study was to examine a direct role for exogenous sublethal flux of superoxide on endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVEC) were exposed to superoxide generated by 0.1 mM xanthine and 4 mU/ml xanthine oxidase for 15 min and essential endothelial functions were examined. Superoxide dismutase and/or catalase was used as scavenger for O2radical dot/H2O2 to determine the key culprit. HUVEC detachment was determined by neutral red uptake and apoptosis by annexin V binding. Inflammation was estimated by IL-8 mRNA expression and cellular adhesion molecules (CAM). eNOS and iNOS message and eNOS protein served as an indirect measure for NO. Procoagulable state was evaluated by estimating the intracellular tissue factor. Activation of endothelial NADPH oxidase was determined by lucigenin chemiluminescence. Sublethal superoxide dose evoked: (1) proinflammatory state manifested by increased IL-8 mRNA expression and CAM on the endothelial surface, (2) HUVEC apoptosis and activated endothelial NADPH oxidase, (3) increase in intracellular tissue factor, and (4) decrease in eNOS mRNA and protein and up-regulation of iNOS mRNA. We conclude that extracellular low flux of superoxide exhibits pleiotropic characteristics, triggering activation/dysfunction of endothelial cells.

Section snippets

Endothelial cell culture

HUVEC were cultured as described by Jaffe et al. [14] with slight modifications according to Lanir et al. [15]. Cells isolated from the cord vein were plated on fibronectin-coated tissue culture flasks at 100% humidity and 5% CO2 and allowed to adhere overnight at 37°C with medium changes every 2 days. The medium used was M-199 (Biological Industries, Beit Haemek, Israel) containing 10% heat-inactivated fetal calf serum (FCS: Biological Industries), 8% heat-inactivated human serum, 35 μg/ml

Superoxide and H2O2 production by the X/XO system: the effect of the antioxidants SOD and catalase

Superoxide production from X/XO was quantitated over a 15-min period, at 37°C at pH 7.4 using the cytochrome c reduction assay as described under Materials and methods. As shown by representative curves (Fig. 1A), superoxide production increased in a time-dependent manner with a yield of ∼15–20 nmol/ml/15 min. Addition of catalase (66 mU/ml) to the system did not affect superoxide production as opposed to SOD (85 U/ml), which fully inhibited the superoxide production.

H2O2 production by the X/XO

Discussion

This study demonstrates a critical direct role for exogenously produced superoxide in a sublethal dose in triggering endothelial cell dysfunction/activation. Exogenous sublethal superoxide exhibits pleiotropic effects on endothelial cells such as induction of apoptosis, inflammation, coagulation, and intracellular ROS production. This sublethal dose mimics physiological concentrations of superoxide chronically generated by primed PMNL in the circulation [10], [11], [12], [13]. However, primed

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