Postprandial apoE isoform and conformational changes associated with VLDL lipolysis products modulate monocyte inflammation.

ObjectivePostprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.Methods and resultsWe showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.ConclusionPostprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation

Fasting VLDL lipolysis products attenuate LPS-induced TNFα release.

<p>(A-B) Monocytes treated with fasting or postprandial VLDL lipolysis products and LPS exhibited dose-dependent attenuation of TNFα secretion (n = 6). A: fasting VLDL; B: postprandial VLDL. *P<0.01 from control, #P<0.05 from the same concentration dose without LpL. (C) Postprandial remnants (VLDL rem) attenuate LPS-induced TNFα secretion more than fasting remnants. *P<0.05 from control, #P<0.05 from fasting.</p

Blocking apoE from fasting VLDL lipolysis products reverses the attenuation of LPS-induced TNFα release.

<p>Monocytes were treated with apoE-blocked VLDL lipolysis products from (A) pooled normal (n = 6), (B) apoE3/E3 subjects (n = 3), and (C) apoE4/E4 subjects (n = 3). Blocking fasting samples reversed the attenuated TNFα release, except for apoE4/E4. Different letters represent significant differences between groups; P<0.01 for a vs. b and a vs. c; P<0.05 for b vs. c.</p

Interaction of apoE C-terminal domain is increased by LPS.

<p>EPR spectra of spin-labeled apoE4-264C and E3-like apoE-W264C+Arg112→Ser in the absence (A) and presence (B) of LPS. LPS induces spectral broadening of both apoE4 and the E3-like proteins (inset), but the magnitude of the effect is greater with E3-like protein (B).</p

Differential effects of apoE3 and apoE4 on LPS-induced cytokine expression and secretion.

<p>ApoE3 or apoE4 were complexed with extruded DMPC vesicles, then LPS was added for treatment of THP-1 cells. (A) TNFα and IL-1β gene expression and (B) TNFα protein secretion were quantified and normalized to the LPS+DMPC control for each treatment, (n = 5). *P<0.05.</p

Blocking apoB-100, apoCII, and apoCIII on VLDL lipolysis products reverses the attenuation of LPS-induced TNFα secretion.

<p>Monocytes were treated with (A) apoB-100, (B) apoCII, or (C) apoCIII-blocked fasting and postprandial VLDL lipolysis products from pooled normal subjects (n = 3). Blocking each apolipoprotein from both fasting and postprandial samples reversed the attenuated TNFα release. Different letters represent significant differences between groups; P<0.01 for a vs. b and c; P<0.05 for a vs. d.</p