Involvement of CYP 2C9 in mediating the proinflammatory effects of linoleic acid in vascular endothelial cells.
OBJECTIVE
Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD).


METHODS
The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry.


RESULTS
Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations.


CONCLUSIONS
Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.
Viswanathan
Saraswathi
S
Molecular and Cell Nutrition Laboratory, College of Agriculture, University of Kentucky, Lexington, KY 40546-0215, USA.
Hammock
Bruce D
BD
Newman
John W
JW
Meerarani
Purushothaman
P
Toborek
Michal
M
Hennig
Bernhard
B
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D004306:Dose-Response Relationship, Immunologic; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005098:Exotoxins / Q000378:metabolism / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D005786:Gene Expression Regulation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D018836:Inflammation Mediators / Q000378:metabolism*; D019787:Linoleic Acid / Q000378:metabolism* / Q000494:pharmacology*; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013229:Stearic Acids / Q000378:metabolism / Q000494:pharmacology; D013552:Swine; D018808:Transcription Factor AP-1 / Q000187:drug effects / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
C120038:9,10-dihydroxy-12-octadecenoic acid; D005098:Exotoxins; D007166:Immunosuppressive Agents; D018836:Inflammation Mediators; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013229:Stearic Acids; D018808:Transcription Factor AP-1; C018579:leukotoxin; D019787:Linoleic Acid; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D005978:Glutathione