Production of Free Radicals and Oxygen Consumption by Primary Equine Endothelial Cells During Anoxia-Reoxygenation



Geoffroy de la Rebière de Pouyade*, 1, 2, Alexandra Salciccia 1, 2, Justine Ceusters 2, Ginette Deby-Dupont 2, Didier Serteyn 1, 2, Ange Mouithys-Mickalad*, 2
1 Department of Clinical Sciences, Equine Clinic, Faculty of Veterinary Medicine B41, University of Liège, Sart Tilman, 4000 Liège, Belgium
2 Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, Sart Tilman, 4000 Liège, Belgium


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© de la Rebière de Pouyade et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http: //creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to these authors at the Center for Oxygen Research and Development, Institute of Chemistry B6a, University of Liège, Sart Tilman, 4000 Liège, Belgium; Tel: +32 4-366-33-66; Fax: +32 4-366-28-66; E-mails: G.delaRebieredePouyade@ulg.ac.be; amouithys@ulg.ac.be


Abstract

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced α-keto-γ-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significantly decreased with the number of A/R cycles, and POBN-ESR spectra were specific of adducts formed in the cells from superoxide anion. After a one-hour A/R cycle, high intensity DMPO-ESR spectra were observed and assigned to superoxide anion trapping; the GC results confirmed an important production of ROS compared to normoxic cells. These results show that A/R induces mitochondrial alterations in endothelial cells, and strongly stimulates their oxidative activity as demonstrated by ESR and GC methods.

Keywords: Equine endothelial cell, Anoxia/Re-oxygenation, High-resolution Oxygraphy, Gas Chromatography, Electron Spin Resonance, Reactive Oxygen Species, Free radicals.