Endotoxin-induced HIF-1alpha stabilisation in equine endothelial cells: synergistic action with hypoxia.

Abstract

OBJECTIVE AND DESIGN: Hypoxia may enhance the deleterious effects of lipopolysaccharide (LPS) in the endotoxaemic horse. This study has examined some of the actions of LPS and hypoxia, alone and in combination, on cultured equine digital vein endothelial cells (EDVEC) and the signalling molecules involved. METHODS: EDVEC were exposed to LPS, 5% O(2) and LPS then 5% O(2) for up to 24 h. HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability were assessed by immunoblotting, measurement of myeloperoxidase and movement of FITC-dextran, respectively. Pharmacological inhibitors were used to assess the roles of p38 MAPK and HIF-1alpha. RESULTS: LPS and hypoxia significantly increased HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability and the effects of the two stimuli in combination on HIF-1alpha stabilisation and neutrophil adhesion were more than additive. The effect of LPS, but not 5% O(2), on neutrophil adherence required activation of p38 MAPK, whereas EDVEC permeability in response to both stimuli was dependent on p38 MAPK and HIF-1alpha. CONCLUSIONS: Exposure of EDVEC to LPS prior to induction of hypoxia up-regulates responses that may enhance LPS-induced tissue damage in the endotoxaemic horse. Inhibitors of p38 MAPK or HIF-1alpha could reduce such unwanted effects.