Original ContributionExogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures
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
References (51)
Endothelial cell activation
Kidney Int.
(1998)Atherosclerosis is an inflammatory disease
Am. Heart J.
(1999)- et al.
Lead exposure raises superoxide and hydrogen peroxide in human endothelial and vascular smooth muscle cells
Kidney Int.
(2004) - et al.
Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide
J. Biol. Chem.
(2001) - et al.
The oxidants hypochlorite and hydrogen peroxide induce distinct patterns of acute lung injury
Biochim. Biophys. Acta
(2004) - et al.
Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension
Am. J. Hypertens.
(1998) - et al.
Reactivity patterns of antiphospholipid antibodies and endothelial cells: effect of antiendothelial antibodies on cell migration
J. Lab. Clin. Med.
(1998) - et al.
Enhanced production and action of cyclic ADP-ribose during oxidative stress in small bovine coronary arterial smooth muscle
Microvasc. Res.
(2004) - et al.
Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V
Blood
(1996) - et al.
Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells
Free Radic. Biol. Med.
(1997)