Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation

Abstract

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.

Fig. 1.

PUFAs from triglyceride-rich lipoprotein (TGRL) incubated with or without lipoprotein lipase (LpL). Human postprandial VLDL (A), chylomicron (CM) (B), or TGRL (C) [2.5 mg triglyceride (TG) for all treatments] with or without LpL were used for lipid extraction and oxidized lipids measurement. For LpL-dependent lipolysis, LpL was preincubated with CM, VLDL, or TGRL for 30 min at 37°C, and the reaction was stopped by transferring the tubes on ice prior to sample extraction and FA analysis. The concentration of linoleic acid (LA) and arachidonic acid (AA), as well as the n-3 FAs α-linoleic acid (ALA), eicosapentenoic acid (EPA), and docosahexaenoic acid (DHA) in VLDL, CM, and TGRL increased with LpL treatment. ^a Lipids were pseudo-quantified and adjusted by relative response factors to estimate their concentrations and allow consistent graphical representation of these data. Data are mean ± SEM (n = 3).

Fig. 2.

Oxidized lipids from VLDL incubated with or without LpL. Concentrations of oxylipids were increased by LpL treatment. Human postprandial VLDLs (2.5 mg TG) with or without LpL were used for lipid extraction and oxidized lipids measurement. For LpL-dependent VLDL lipolysis, LpL was preincubated with VLDL for 30 min at 37°C, and the reaction was stopped by transferring the tubes on ice prior to sample extraction. The LA oxidation products (A) epoxy octadecenoic acid (EpOME), dihydroxy octadecanoic acid (DHOME), hydroxy ocatadecadienoic acid (HODE), and oxo octadecanoic acid (oxo-ODE), as well as the AA oxidation products (B) epoxy eicosatrienoic acid (EET), dihydroxy eicosatrienoic acid (DHET), hydroxy eicosatetraenoic acid (HETE), and oxo eicosatetraenoic acid (oxo-ETE) were determined. Data are mean ± SEM (n = 3).

Fig. 3.

VLDL and its LpL lipolysis-derived fractions increase endothelial cell TNFα production (A) and intracellular adhesion molecule (ICAM) expression (B). TNFα level and ICAM expression on human aortic endothelial cells were measured by ELISA. Endothelial cell cultures in 12-well plates were treated for 2 h with individual lipids isolated from VLDL (50 mg/dl TG) with or without 30 min incubation with LpL (2 U/ml). Each fraction was isolated from VLDL, evaporated, redissolved, and delivered to cells in a PBS vehicle (5 μl). TNFα level or ICAM expression stimulated with TNFα (10 ng/ml, 16 h) was measured. Excess FFA released from lipolysis increased TNFα expression and TNF-induced ICAM expression on HAECs. Data are mean ± SEM (n = 6). CTRL, control; FFA, free fatty acid; PL, phospholipid; CE, cholesteryl ester; TG, triglyceride; FC, free cholesterol; DG, diglyceride; MG, monoacylglycerol.

Fig. 4.

Effects of lipid fractions isolated from VLDL on reactive oxygen species (ROS) production in HAECs. Each lipid fraction was isolated from VLDL (50 mg/dl TG), evaporated, redissolved, and delivered to cells in a PBS vehicle (5 μl). HAECs were preincubated for 30 min with the H₂O₂-sensitive fluorescence probe DCF-AM (10 μM), followed by incubation for 2 h with individual lipids isolated from VLDL and 30 min incubation with LpL (2 U/ml). Fluorescence intensity of cells was measured with a fluorescence microplate reader. Fluorescence distribution of DCF-AM oxidation was expressed as fluorescence units (FU) and expressed as the mean ± SEM (n = 6). * P < 0.01. CTRL, control; FFA, free fatty acid; PL, phospholipid; CE, cholesteryl ester; TG, triglyceride; FC, free cholesterol; DG, diglyceride; MG, monoacylglycerol.

Fig. 5.

Effects of FFA isolated from VLDL (V-FFA) and VLDL lipolysis (V + L-FFA) on ROS production in HAECs in the absence or presence of enzyme inhibitors. Each lipid fraction was isolated from VLDL (50 mg/dl TG), evaporated, redissolved, and delivered to cells in a PBS vehicle (5 μl). HAECs were preincubated for 30 min with the fluorescence probe DCF-AM (10 μM), followed by incubation for 2 h with FFA isolated from VLDL with or without 30 min incubation with LpL (2 U/ml) in the absence or presence of allopurinol (FFA-AP) (100 μM), apocynin (FFA-AC) (100 μM), sulfaphenazole (FFA-SP) (10 μM), or DPI (FFA-DPI) (50 μM). Fluorescence intensity of cells was measured with a fluorescence microplate reader. Fluorescence distribution of DCF-AM oxidation was expressed as fluorescence units (FU) and expressed as the mean ± SEM (n = 6). Significant differences in means were determined by 2-tailed Student's t-test (* P < 0.05 vs. V-FFA; **P < 0.05 vs. V+L-FFA).