Involvement of CYP 2C9 in Mediating the Proinflammatory Effects of Linoleic Acid in Vascular Endothelial Cells

Involvement of CYP 2C9 in Mediating the Proinflammatory Effects of Linoleic Acid in Vascular Endothelial Cells

Saraswathi Viswanathan, PhD, Bruce D. Hammock, PhD, John W. Newman, PhD, Purushothaman Meerarani, PhD, Michal Toborek, MD, PhD and Bernhard Hennig, PhD, FACN

Molecular and Cell Nutrition Laboratory, College of Agriculture (S.V., P.M., B.H.), USA
Department of Surgery (M.T.), University of Kentucky, Lexington, KY, 40546-0215, USA
Department of Entomology and UC Cancer Center (B.D.H., J.W.N.), University of California, Davis, CA, USA

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Fig. 1. Effect of linoleic acid (LA) on CYP 2C9 messenger RNA levels in human endothelial cells as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Human umbilical vein endothelial cells were exposed to LA for six hours. The amplified PCR products were visualized using phosphor-imaging technology. Lane 1, control; lane 2, LA (90 µM). The values are presented as mean ± SEM of three sets of experiments. *Significantly different from control values.

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Fig. 2. Effect of sulfaphenazole on cellular oxidative stress as measured by DCFH oxidation (A) and cellular glutathione status (B) in endothelial cells exposed to linoleic acid (LA). For the measurement of DCF fluorescence, cells were exposed to 90 µM LA in the presence or absence of 10 µM sulfaphenazole (SP) for three hours. Fluorescence intensity was measured using a fluorescent plate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 ± 5 nm. For the measurement of intracellular glutathione, cells were treated with 90 µM LA and/or SP for six hours. The values are presented as mean ± SEM of three sets of experiments. *Significantly different from control values. ^#Significantly different from LA group.

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Fig. 3. Effect of MnTMPyP on cellular oxidative stress (DCFH oxidation) (Fig. 3A) and superoxide generation (Figure 3B) in endothelial cells exposed to linoleic acid (LA). Cells were exposed to 90 µM LA in the presence or absence of 10 µM MnTMPyP for three hours. Cells were loaded with either DCFH-DA or DHE (10 µM) for the last 30 minutes of the treatment period. After staining, cells were washed with HEPES buffer (pH 7.4), and the fluorescence intensity was measured using a fluorescent plate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 ± 5 nm for DCF and at an excitation wavelength of 520 ± 5 nm and an emission wavelength of 620 ± 5 nm for DHE. The values are presented as means ± SEM of three sets of experiments. *Significantly different from control values. ^#Significantly different from LA group.

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Fig. 4. Effect of 10 µM sulfaphenazole (SP) on linoleic acid (LA)-mediated nuclear translocation of AP-1 in porcine pulmonary artery endothelial cells. Confluent monolayers were treated with 90 µM LA in the presence or absence of SP for six hours. Lane 1, control; lane 2, SP (10 µM); lane 3, LA (90 µM); and lane 4, LA (90 µM) + SP (10 µM). The values are presented as means ± SEM of three separate experiments. *Significantly different from control values. ^#Significantly different from LA group.

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Fig. 5. Effect of leukotoxin (LTX) and leukotoxin diol (LTD) on cellular oxidative stress (DCFH oxidation) in endothelial cells. Cells were exposed to LTX and LTD (60 and 90 µM) for three hours. Fluorescence intensity was measured at an excitation wavelength of 490 nm and an emission wavelength of 525 ± 5 nm. Values are mean ± SEM of three separate experiments. *Significantly different from control values.

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Fig. 6. Effect of leukotoxin (LTX) and leukotoxin diol (LTD) on the activation of NF- ${kappa}$ B (A) and AP-1 (B) in porcine pulmonary artery endothelial cells. Confluent monolayers were treated with LTX (60 and 90 µM) and LTD (60 and 90 µM) for six hours. Fig. 5A: lane 1, control; lane 2, LTX (60 µM); lane 3, LTX (90 µM); lane 4, LTD (60 µM); lane 5, LTD (90 µM) and lane 6, LTX (90 µM) supershift (p65). Fig. 5B: lane 1, control; lane 2, LTX (60 µM); lane 3, LTX (90 µM); lane 4, LTD (60 µM); lane 5, LTD (90 µM) and lane 6, LTX (90 µM) supershift (c-jun). Values are mean ± SEM of three separate experiments. *Significantly different from control values.

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Fig. 7. Endothelial cell-derived linoleic acid metabolites. Cells were cultured in the presence of linoleic acid (90 µM; upper trace) for 24 hours, and media concentrations of linoleate-derived epoxides and diols were 3.2 ± 0.5 nM and 90 ± 15 nM, respectively. These metabolites were present at <0.5 and 10 ± 5 nM, respectively without supplemental linoleic acid. Traces of epoxy and dihydroxy arachidonates were also observed in linoleic acid-treated cultures (data not shown). Results are from triplicate analyses of 6 mL culture media aliquots analyzed by LC/MS/MS.

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Fig. 8. Proposed model for the mechanism of linoleic acid (LA)-mediated endothelial cell activation. LA treatment results in CYP 2C9 activation and production of superoxide radicals as well as depletion of glutathione in endothelial cells. The increased oxidative stress results in the activation of oxidative stress sensitive transcription factors such as NF- ${kappa}$ B and AP-1, leading to endothelial cell activation. Sulfaphenazole, a specific inhibitor of CYP 2C9 suppresses the oxidative stress caused by LA treatment. The formation of leukotoxin and leukotoxin diol under physiological condition may help in the fatty acid detoxification process.