Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin.
OBJECTIVE
To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs).


METHODS
EDVECs obtained from forelimb digital veins of 7 healthy adult horses.


METHODS
EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined.


RESULTS
LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner.


CONCLUSIONS
LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.
Menzies-Gow
Nicola J
NJ
Department of Veterinary Clinical Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.
Bailey
Simon R
SR
Berhane
Yoel
Y
Brooks
Andrew C
AC
Elliott
Jonathan
J
D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011464:Epoprostenol / Q000378:metabolism; D005552:Forelimb; D006734:Horse Diseases / Q000139:chemically induced* / Q000201:enzymology / Q000378:metabolism*; D006736:Horses; D007052:Ibuprofen / Q000494:pharmacology; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D013928:Thromboxane A2 / Q000378:metabolism
D019332:Endothelin-1; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D013928:Thromboxane A2; D003513:Cycloheximide; D011464:Epoprostenol; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester; D007052:Ibuprofen