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. 2017 Jul 21;121(3):244-257.
doi: 10.1161/CIRCRESAHA.116.310308. Epub 2017 May 18.

Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell-Mediated Generation of LXA4

Yunbo Ke  1 , Noureddine Zebda  1 , Olga Oskolkova  1 , Taras Afonyushkin  1 , Evgeny Berdyshev  1 , Yufeng Tian  1 , Fanyong Meng  1 , Nicolene Sarich  1 , Valery N Bochkov  1 , Ji Ming Wang  1 , Anna A Birukova  1 , Konstantin G Birukov  2
Affiliations

Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell-Mediated Generation of LXA4

Yunbo Ke et al. Circ Res. .

Abstract

Rationale: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling.

Objective: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4).

Methods and results: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-α in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-α and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA-induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2-/- (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX.

Conclusions: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.

Keywords: endothelial cells; inflammation; lipoxins; lung injury; phospholipids; pulmonary circulation.

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Figures

Figure 1
Figure 1. Inhibition of FPR1-3 receptors does not affect barrier-enhancing effects of OxPAPC on human pulmonary EC
A - Expression of FPR1, FPR2/ALX and FPR3 mRNA in HPAEC was evaluated by qRT-PCR. PCR products corresponding to FPR1, FPR2/ALX and FPR3 were resolved by agarose gel electrophoresis and visualized by gel staining with Ethidium Bromide (red) and SYBR green (green) B - HPAEC plated on microelectrodes were treated with non-specific RNA oligomers (nsRNA) or gene-specific siRNA to FPR1, FPR2/ALX and FPR3, respectively (50 nM, 72 hrs). TER measurements were performed following stimulation with OxPAPC (15 μg/ml, marked by arrow). C and D - TER measurements of HPAEC monolayers preincubated with FPR receptor peptide inhibitors: C - WRW4, 2.5 or 5 μM, or: D - Boc-FLFLF, 10, 20 or 40 μM, 1 hr prior to OxPAPC treatment (marked by arrow). The TER curves represent pooled data from three independent experiments.
Figure 2
Figure 2. FPR2/ALX knockdown does not affect barrier protective effect of OxPAPC against thrombin-induced permeability
Pulmonary EC transfected with non-specific or FPR1, FPR2/ALX, or FPR3 specific siRNAs were treated with thrombin (0.5 U/ml), with or without OxPAPC pretreatment (15 μg/ml, 30 min). A - Analysis of EC permeability for macromolecules using FITC-labeled avidin as a tracer; n=6, *P < 0.01 vs. thrombin alone. B - MLC di-phosphorylation was evaluated using phospho-MLC specific antibody in control, thrombin, or OxPAPC treated EC. C - Effects of OxPAPC and LXA4 (100 nM) on thrombin-induced MLC phosphorylation were evaluated by Western blotting. Probing for β-tubulin was used as a normalization control. Numerical data depict results of quantitative densitometry; n=4; p<0.05 vs. thrombin alone.
Figure 3
Figure 3. Effect of OxPAPC and LXA4 on TNFα-induced inflammatory activation of human pulmonary EC
Cells were treated with TNFα (20 ng/ml, 6 hrs) alone or pretreated with OxPAPC (15 μg/ml) or LXA4 (100 nM) for 30 min. A - IκBα degradation, ICAM1 and VCAM1 expression were analyzed by Western blotting. Probing for β-tubulin was used as a normalization control. Numerical data depict results of quantitative densitometry; n=4; p<0.05 vs. TNFα alone. B - Expression of ICAM1 mRNA in control and stimulated HPAEC was evaluated by qRT-PCR; n=3, *P < 0.05 vs. TNFα alone. C - The level of soluble ICAM1 (sICAM1) in EC conditioned medium after stimulations was measured using ELISA assay; n=5, *P < 0.05 vs. TNFα alone.
Figure 4
Figure 4. Role of FPR2/ALX signaling in anti-inflammatory and barrier protective effects of OxPAPC on pulmonary EC
Cells were treated with TNFα (20 ng/ml) alone or pretreated with OxPAPC (15 μg/ml, 30 min). A - Effect of siRNA-induced FPR1, FPR2/ALX or FPR3 knockdown on EC permeability for macromolecules; n=3, *P < 0.05. B - Visualization of FITC-avidin accumulation underneath EC monolayers reflecting TNFα-induced EC barrier dysfunction. Protective effects of OxPAPC were suppressed by FPR2/ALX inhibitor WRW4 (5 μM, 1 hr prior to OxPAPC). Bar = 20 μm. Bar graph depicts quantitative analysis of FITC-avidin fluorescence in control and stimulated EC monolayers; n=6, *P < 0.05. C - TER measurements in HPAEC monolayers preincubated with WRW4 followed by TNFα challenge (marked by second arrow) with or without OxPAPC pretreatment (marked by first arrow). Bar graph depicts TER measurements at the time point of maximal response indicated by dotted line; n=6, *P < 0.05. D - Effect of FPR2/ALX inhibitor on the changes of EC barrier integrity caused by TNFα and OxPAPC. F-actin was visualized by staining with Texas Red phalloidin (red); adherens junctions were visualized by staining for VE-cadherin (green). Paracellular gaps are marked by arrows. Bar = 10 μm. Bar graph represents results of quantitative analysis of paracellular gap formation; n=4, * p<0.05.
Figure 5
Figure 5. Role of FPR2/ALX signaling in anti-inflammatory effects of OxPAPC and LXA4
Cells were treated with TNFα (20 ng/ml) alone or pretreated with OxPAPC (15 μg/ml) or LXA4 (100 nM) for 30 min. A - Effect of FPR2/ALX inhibitor WRW4 on IκBα degradation, ICAM1 and VCAM1 expression. Probing for β-tubulin was used as a normalization control. B - Effect of siRNA-induced FPR2/ALX knockdown on VCAM1 expression. Numerical data depict results of quantitative densitometry; n=3; * p<0.05. C - Effect of FPR2/ALX inhibitor on sICAM1 release in culture medium evaluated by ELISA assay; n=5, *P < 0.05.
Figure 6
Figure 6. Analysis of OxPAPC-induced LXA4 generation
EC were treated with OxPAPC (15 μg/ml), DMPC (15 μg/ml), OxPAPC + LPS (200 ng/ml), or DMPC + LPS. A – ELISA assay: time course of LXA4 generation by EC treated with OxPAPC, DMPC, or vehicle; n=3, *P < 0.05 vs. vehicle. B - LXA4 generation by EC challenged with LPS (1 hr) and post-treated with OxPAPC (6 or 24 hrs), DMPC (6 hrs), or vehicle; n=4, *P < 0.05 vs. vehicle. C - Generation of LXA4 by EC and in EC-PMN co-culture. Similar levels of LXA4 were detected in EC cultured alone and in EC co-cultured with PMN; n=4, ND – no difference. D - Increased LXA4 levels in lung tissue of LPS-challenged mice after 5-hrs post-treatment with OxPAPC; n=3, *P < 0.05. E - Mass spectrometry analysis of LXA4 generation by cultured EC. Cells were treated with OxPAPC (1–24 hrs), DMPC (1 hr) or LXA4 (1 hr) as a positive control. Culture medium with addition of OxPAPC, DMPC, or LXA4 without exposure to the cells was used as an additional control. F - Mass spectrometry analysis of LXA4 in the conditioned medium of EC stimulated with LPS, LPS+OxPAPC, or LPS+DMPC. Cells were incubated with agonists for 6 or 24 hrs. LXA4 was detected in the conditioned medium; n=3, *P < 0.05 vs. LPS alone.
Figure 7
Figure 7. Analysis of OxPAPC-induced LXA4 generation
EC were preincubated with 5-LO or 15-LO inhibitor for 1 hr followed by TNFα challenge (20 ng/ml, marked by second arrow) with or without OxPAPC pre-treatment (15 μg/ml, marked by first arrow). A - TER measurements reflecting changes in EC permeability were performed over 25-hr time period. B – IL-8 accumulation in EC conditioned medium after stimulations was measured using ELISA assay. C – EC pretreated with sPLA2, 15-LO or 5-LO inhibitors were incubated with OxPAPC or DMPC (15 μg/ml), and LXA4 accumulation in conditioned medium was measured by mass spectrometry; n=3, *P < 0.05; ND – no difference.
Figure 8
Figure 8. Role of FPR signaling in protective effects of OxPAPC in the model of LPS-induced lung injury
A - Intravenous injection of OxPAPC (1.5 mg/kg) with our without FPR2/ALX inhibitor WRW4 (20 μM, 1 hr prior to OxPAPC) was performed 5 hrs after LPS instillation (0.7 mg/kg, i.t.). BAL cell count and protein content were measured after 24 hrs of LPS challenge; n=5, *P < 0.05. B - BAL cell count and protein content in mFPR−/− mice and matching controls treated with LPS and LPS+OxPAPC; n=4, *P < 0.05. C - Evans Blue accumulation in the lung parenchyma. D - ICAM1 expression in lung tissue samples of control and mFPR-/- mice after LPS or LPS+OxPAPC challenge. E - ELISA assay of TNFα and sICAM1 levels in BAL samples; n=4, *P < 0.05.
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