Endotoxin-induced HIF-1alpha stabilisation in equine endothelial cells: synergistic action with hypoxia.
OBJECTIVE
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.
Department of Veterinary Basic Sciences, The Royal Veterinary College, North Mymms, Hertfordshire, UK. abrooks@rvc.ac.uk
A C
AC
Brooks
N
N
Menzies-Gow
S R
SR
Bailey
F M
FM
Cunningham
J
J
Elliott
D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D019446:Endotoxemia / Q000378:metabolism / Q000662:veterinary*; D005260:Female; D006734:Horse Diseases / Q000378:metabolism*; D006736:Horses; D000860:Hypoxia / Q000378:metabolism / Q000662:veterinary*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D009504:Neutrophils / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000032:analysis
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D048051:p38 Mitogen-Activated Protein Kinases