Oleoyl-Lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species

<div><p>Previously we reported modulation of endothelial prostacyclin and interleukin-8 production, cyclooxygenase-2 expression and vasorelaxation by oleoyl- lysophosphatidylcholine (LPC 18:1). In the present study, we examined the impact of this LPC on nitric oxide (NO) bioavailability in vascular endothelial EA.hy926 cells. Basal NO formation in these cells was decreased by LPC 18:1. This was accompanied with a partial disruption of the active endothelial nitric oxide synthase (eNOS)- dimer, leading to eNOS uncoupling and increased formation of reactive oxygen species (ROS). The LPC 18:1-induced ROS formation was attenuated by the superoxide scavenger Tiron, as well as by the pharmacological inhibitors of eNOS, NADPH oxidases, flavin-containing enzymes and superoxide dismutase (SOD). Intracellular ROS-formation was most prominent in mitochondria, less pronounced in cytosol and undetectable in endoplasmic reticulum. Importantly, Tiron completely prevented the LPC 18:1-induced decrease in NO bioavailability in EA.hy926 cells. The importance of the discovered findings for more in vivo like situations was analyzed by organ bath experiments in mouse aortic rings. LPC 18:1 attenuated the acetylcholine-induced, endothelium dependent vasorelaxation and massively decreased NO bioavailability. We conclude that LPC 18:1 induces eNOS uncoupling and unspecific superoxide production. This results in NO scavenging by ROS, a limited endothelial NO bioavailability and impaired vascular function.</p></div

LPC 18:1 induces intracellular and extracellular superoxide production.

<p>(A) ROS levels were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113443#pone-0113443-g002" target="_blank">Figure 2</a> after inhibition of SOD with 20 µM DETCA. (B) Cells plated in 96-well dishes were incubated with 15 µM DHE in the presence of 20 µM DETCA at 37°C for 15 min. Thereafter, cells were exposed to 60 µM LPC 18:1 or PBS (vehicle) in PBS containing 5% FBS at 37°C for 15 min, followed by fluorometric superoxide quantification. (C) Cells grown on a glass coverslip were treated as in B followed by fluorescent microscopy. (D) Amplex Red Assay was performed in cells exposed to 10 µM LPC 18:1 or PBS in the presence or absence of PEG-catalase (300 U/ml) or PEG-SOD (75 U/ml) in HEPES-buffered Tyrode's solution without FBS at 37°C for 15 min. The presented values are catalase-sensitive values obtained by subtraction of values obtained in the presence of catalase from those in the absence of catalase. The values shown are mean ± SEM of 3 independent experiments performed in triplicates and analyzed by one-way ANOVA with Tukey's post-hoc test (A,D) or unpaired t-test (B,C). If otherwise not indicated, asterisks show significance compared to PBS control; *p<0,05, ***p<0,001.</p

LPC 18:1 limits NO bioavailability in cells and mouse aortic segments.

<p>(A) Nitrite levels in cell media of LPC 18:1- or PBS-treated cells were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113443#pone-0113443-g001" target="_blank">Figure 1</a> A in the absence or presence of 100 µM Tiron. (B) Mouse aortic rings (5 for each condition) were incubated with 10 µM LPC 18:1 or PBS in the absence of FBS for 15 min followed by NE preconstriction and relaxation to cumulative addition of Ach. Relaxation values are expressed as a percentage of the initial NE-induced contraction. (C) Mouse aortic rings were treated with 10 µM LPC 18:1 or PBS in the absence of FBS for 15 min and preconstricted with phenylephrine to 10% of maximal contraction followed by addition of 300 µM L-NA (eNOS inhibitor). The ratio (L-NA ratio) of constriction achieved after and before addition of L-NA is shown. (D) Mouse aortic rings were treated with 10 µM LPC 18:1 or PBS in PSS without FBS for 15 min. Nitrite levels determined in the buffer (PSS) post incubation are shown. (E) Cells were treated with 10 µM LPC 18:1 (in medium without FBS) or with 60 µM LPC 18:1 or PBS (both in medium with 5% FBS) for 15 minutes. Nitrite levels determined in the media after treatment are shown. (F) ROS species were measured with H₂DCFDA dye (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113443#pone-0113443-g002" target="_blank">Figure 2</a>) after exposure of cells to 10 µM LPC 18:1 (in medium without FBS) or to 60 µM LPC 18:1 or PBS (both in medium with 5% FBS) for 15 minutes. Results are mean ±SEM of 3 independent experiments done in triplicate and analyzed by one-way ANOVA with Tukey's post-hoc test (A, E, F) or by two-way ANOVA with Bonferroni post-hoc test (B) or unpaired t-test (C, D). If otherwise not indicated, asterisks show significance compared to PBS control; *p<0,05, **p<0,01, ***p<0,001, ns –not significant.</p

LPC 18:1 induced ROS localizes mainly in mitochondria and cytosol.

<p>Twenty four hours after transfection of EA.hy926 cells with plasmids encoding either (A) mitochondria- or (B) ER- targeted RFP, cells were labeled with H₂DCFDA dye and exposed to 60 µM LPC 18:1 or PBS (vehicle) in PBS containing 5% FBS at 37°C for 15 min. Fluorescence was assessed by confocal microscopy using mitochondrial- and endoplasmic reticulum-specific RFP marker (red) and a H₂DCFDA ROS marker (green). Colocalization (yellow) was achieved by merging RFP and ROS signals (merge). Results are representative images of two experiments performed in triplicates.</p