Effects of electronegative VLDL on endothelium damage in metabolic syndrome

Abstract

Objective: Biochemical heterogeneity governs functional disparities among lipoproteins. We examined charge-defined VLDL subfractions in metabolic syndrome (MetS) to determine whether their increased electronegativity is associated with increased cytotoxicity and whether high concentrations of highly electronegative subfractions render VLDL harmful to the vascular endothelium.

Research design and methods: Plasma VLDL of normal individuals (control subjects) (n = 13) and of those with MetS (n = 13) was resolved into subfractions with increasing negative charge (V1-V5) by anion-exchange chromatography. Human aortic endothelial cells were treated with V1-V5 or unfractionated VLDL.

Results: Compared with the control subjects, individuals with MetS had a significantly higher percentage of V5 VLDL (V5/VLDL%) (34 ± 20 vs. 39 ± 11%, respectively; P < 0.05) and plasma V5 concentration ([V5]) (5.5 ± 4.4 vs. 15.2 ± 8.5 mg/dL, respectively; P < 0.001). Apolipoprotein (apo)B100 levels decreased and apoC levels increased from V1 to V5, indicating that V5 is apoC-rich VLDL. Regression analyses of all 26 individuals showed that [V5] was positively correlated with total cholesterol (P = 0.016), triglyceride (P < 0.000001), and V5/VLDL% (P = 0.002). Fasting plasma glucose, but not waist circumference, exhibited a positive trend (P = 0.058); plasma HDL cholesterol exhibited a weak inverse trend (P = 0.138). V5 (10 μg/mL) induced apoptosis in ~50% of endothelial cells in 24 h. V5 was the most rapidly (<15 min) internalized subfraction and induced the production of reactive oxygen species (ROS) in endothelial cells after 20 min. Unfractionated MetS VLDL, but not control VLDL, also induced ROS production and endothelial cell apoptosis.

Conclusions: In populations with increased risk of diabetes, the vascular endothelium is constantly exposed to VLDL that contains a high proportion of V5. The potential impact of V5-rich VLDL warrants further investigation.

Figure 1

VLDL subfractions in control (Ctrl) and metabolic syndrome (MetS) subjects. A: Distribution of V1–V5 (labeled as 1–5) in VLDL of control and MetS subjects resolved by anion-exchange chromatography (representative of 13 subjects for each group). mAU, absorbance at 280 nm. B: V5/VLDL% in control and MetS subjects. C: [V5] in control and MetS subjects. D: SDS-PAGE gel of V1–V5 from VLDL of MetS subjects showing relative amounts of apoB100, apoE, and apoC. E: Nuclear staining of human aortic endothelial cells exposed to 10 μg/mL V1–V5 and 50 μg/mL L1–L5 viewed under epifluorescence microscopy. Condensed or fragmented nuclei indicate cells undergoing apoptosis. Images are representative of six experiments with V1–V5 and L1–L5.The percentage of cells undergoing apoptosis was evaluated in six samples. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS; †P < 0.05 between V5 and L5. (A high-quality color representation of this figure is available in the online issue.)

Figure 2

Linear regression analyses of correlations between [V5] and the components of MetS criteria (i.e., FPG, waist circumference [WC], HDL cholesterol, and triglyceride [TG]), total cholesterol (TC), and V5/VLDL.

Figure 3

Internalization of VLDL particles and ROS induced by VLDL subfractions V1 and V5 in human aortic endothelial cells. A: Internalization of DiI-labeled (red) V1 and V5 at 15 min and 24 h (hr) after incubation with endothelial cells with or without pretreatment with RAP. B: Detection of ROS production (green) after 20 min of exposure to PBS, V1, control (Ctrl) VLDL, ROS inducer (pyocyanin), V5, or MetS VLDL. (A high-quality digital representation of this figure is available in the online issue.)

Figure 4

Differences in proapoptotic potency between control (Ctrl) and MetS VLDL. Nuclear staining of human aortic endothelial cells exposed to 20 μg/mL VLDL from five control (Ctrl1–Ctrl5) subjects, PBS, or 20 μg/mL VLDL from four MetS (MetS1–MetS4) subjects viewed under epifluorescence microscopy. Effects of V1 and V5 from MetS1 and MetS3 were also examined. Normal nuclei appear blue with DAPI staining. Condensed or fragmented nuclei (bright green) indicate cells undergoing apoptosis. (A high-quality digital representation of this figure is available in the online issue.)