Anoxia/Reoxygenation of Equine Endothelial Cells Produce Reactive Oxygen Species: An Oxymetric and Electron Paramagnetic Resonance Investigation

Abstract Title: Anoxia / Reoxygenation of equine endothelial cells produce reactive oxygen species: an oxymetric and electron paramagnetic resonance investigation.

G. de la Rebière de Pouyade 1,2, A. Mouithys-Mickalad 2, A. Salciccia 1,2, J. Ceusters 2, G. Deby-Dupont 2, D. Serteyn 1,2
1-Department of Clinical Sciences, Large Animal Surgery, 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

Take Home Message: Equine endothelial cells (EC) submitted to anoxia/reoxygenation (A/R) produce reactive oxygen species (ROS).

Introduction: Endothelium participates to the regulation of the inflammatory response and ischemia/reperfusion-induced injuries are suspected in laminitis. Herein, we investigated the effect of anoxia/reoxygenation on ROS production by EC.

Materials and Methods: EC cultures were obtained from equine carotid. Detached cells (107) were submitted either to 3 cycles of 20min anoxia followed by reoxygenation in presence of POBN (50mM)/ethanol (2%) mixture as spin trap or to 1h of anoxia followed by reoxygenation and immediate addition of 100mM DMPO. Normoxic cells were used as control (n=3). Anoxia and reoxygenation were monitored by high-resolution respirometry and ROS production was evidenced by electron paramagnetic resonance (EPR).

Results: After A/R cycles, high intensity EPR spectra, assigned to the POBN/•CH(OH)CH3 adducts were observed, but not in normoxic cells (control). After 1h of anoxia followed by reoxygenation, an enhanced production of DMPO-OH adducts was observed compared to control.

Discussion: The EPR spectra were consistent with the trapping of superoxide anion produced by EC. Such a production could derive from either xanthine oxidase activity or mitochondria dysfunction. Production of ROS by EC could be involved in the disruption of the endothelial barrier, the recruitment of neutrophils and participate to the development of laminitis.

Clinical relevance: Drugs (fractionated heparin or pentoxifylline) able to protect endothelium may have therapeutic relevance in preventing laminitis.

Conclusion: ROS from EC submitted to A/R might play a crucial role in the oxidative aspect of laminitis.