Oxylipins in triglyceride-rich lipoproteins of dyslipidemic subjects promote endothelial inflammation following a high fat meal

Abstract

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.

Figure 1

Postprandial TGRL modulates VCAM-1 surface expression in endothelial cells in proportion to an individual’s postprandial change in triglycerides. Human aortic endothelial cells (HAEC) were treated with TNFɑ (0.3 ng/ml) in the presence or absence of postprandial TGRL (10 mg/dl ApoB) for 4 hr, and CAM surface expression quantified by flow cytometry and reported relative to TNFα stimulation alone. (a) VCAM-1 expression across all subjects (black, N = 39) significantly correlated with the change in serum triglycerides in response to the meal. A threshold for change in plasma triglycerides (ΔTG, postprandial - fasting) >120 mg/dl distinguished the capacity of a subject’s TGRL to elicit an increase in VCAM-1 surface expression (inset). (b) There was no such correlation with ICAM-1 expression. (c) Postprandial TGRL from pro-atherogenic subjects (red, n = 5) significantly enhanced VCAM-1 expression compared to anti-atherogenic subjects (blue, n = 5), but did not differ with respect to their effect on ICAM-1 expression.

Figure 2

Network map depicting the fatty acids and metabolites measured in the esterified pool of TGRL. Superimposed in color are the fold change for fatty acids and oxylipins identified as significantly enriched (red) or depleted (blue) postprandially over all subjects. Colored arrows depict significant changes in the ratio of the abundance of the metabolites connected. Black boxes indicate metabolites that were detected but unchanged by the meal. White boxes are metabolites that were part of the analysis but not detected. Significance was determined using a two factor ANOVA comparing fasting to postprandial in the same subjects, and accounting for pro- and anti-atherogenic response. A Benjamini-Hochberg FDR multiple test correction was applied.

Figure 3

Network map depicting the fatty acids and metabolites measured in the non-esterified pool of TGRL. Superimposed in color are the fold change for fatty acids and oxylipins identified as significantly enriched (red) or depleted (blue) postprandially over all subjects. Colored arrows depict significant changes in the ratio of the abundance of the metabolites connected. Black boxes indicate metabolites that were detected but unchanged by the meal. White boxes are metabolites that were part of the analysis but not detected. Significance was determined using a two factor ANOVA comparing fasting to postprandial in the same subjects, and accounting for pro- and anti-atherogenic response. A Benjamini-Hochberg FDR multiple test correction was applied.

Figure 4

sEH-derived diols and LOX-derived alcohols strongly discriminate the response to the meal between pro- and anti-atherogenic subjects. PLS-DA analysis of postprandial TGRL samples characterized as pro- and anti-atherogenic (5 per group), using postprandial oxylipin abundance adjusted by fasting levels in the same individuals (postprandial/ fasting). The scores plot reveals the separation between pro- and anti-atherogenic-TGRL (Q² = 0.32). The PLS-DA model was constructed using all detected oxylipins. The loadings plot displays the 22 oxylipins that contributed significantly to discrimination between the groups (VIP > 1). The size of the circles corresponds to the VIP score. Each oxylipin is color coded by the enzyme from which it is derived.

Figure 5

Postprandial fatty acid and oxylipin abundance in TGRL predicts changes in VCAM-1 surface expression by HAEC. The model was generated iteratively from all oxylipin, fatty acids, and calculated lipogenesis indices, clustered based on postprandial expression levels in TGRL across all subjects. Cluster scores were calculated as a linear combination of abundance of each cluster constituent, and used to fit the measured Johnson normalized VCAM-1 expression. The strongest model for predicting VCAM-1 expression consisted of a combination of two clusters as depicted. Dashed lines = 95% confidence interval, =Pro-atherogenic TGRL and =Anti-atherogenic TGRL; n = 5.

Figure 6

Fasting fatty acid and oxylipin abundance in TGRL predicts changes in VCAM-1 surface expression by HAEC. The model was generated iteratively from all oxylipin, fatty acids, and calculated lipogenesis indices, clustered based on fasting expression levels in TGRL across all subjects. Cluster scores were calculated as a linear combination of abundance of each cluster constituent, and used to fit the measured Johnson normalized VCAM-1 expression. The strongest model for predicting VCAM-1 expression consisted of a combination of three clusters as depicted. Dashed lines = 95% confidence interval, =Pro-atherogenic TGRL and =Anti-atherogenic TGRL; n = 5.

Figure 7

9-HODE and 12,13-DiHOME reduce VCAM-1 surface expression in TNFα-stimulated HAEC. Representative methyl esters of oxylipins identified as contributing significantly to distinguishing a pro- atherogenic phenotype were delivered to TNFα-stimulated HAEC (0.3 ng/ml, 4hrs). 9-HODE and 12,13-DiHOME reduced VCAM-1 surface expression, whereas 19, 20-DiHDoPE did not alter VCAM-1 surface expression relative to TNFα. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001 by one-way ANOVA followed by Dunnet’s post-test. n = 3–4.