Endothelial Lipase Exerts its Anti-Atherogenic Effect through Increased Catabolism of β-VLDLs

Figures

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  Supplementary Fig. 1.. Validation of DiI distribution in lipoproteins WT rabbits fed a normal diet were intravenously injected with fluorescent labeled β-VLDLs (DiI-β-VLDL). Blood samples were withdrawn at 0, 2, 30, 60 and 120 min after DiI-β-VLDL administration. Plasma lipoproteins were isolated using sequential ultracentrifugation. Density fractions contained following lipoproteins: VLDL (d < 1.006 g/mL), LDL (d= 1.006∼1.063 g/ml), and HDL (d= 1.063∼1.21 g/ml). (A) Fluorescent intensity and (B) total cholesterol (TC) were measured. Two rabbits were evaluated for the validation of DiI-β-VLDL stability and representative data are shown.

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  Fig. 1.. Plasma lipids and β-VLDL clearance (A) Plasma total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels of transgenic (Tg) and non-Tg rabbits fed a normal standard and cholesterol-rich diet for 1 week. Data are expressed as mean ± SEM (n = 8–12). *P < 0.05, ***P < 0.001 vs non-Tg rabbits by t-test. (B) β-very-low-density lipoprotein (β-VLDL) clearance. Tg and non-Tg rabbits were intravenously injected with fluorescentlabeled β-VLDLs (DiI-β-VLDL). Blood samples were withdrawn at 2, 5, 15, 30, and 60 min after DiI-β-VLDL administration. Plasma fluorescent intensity was measured by a fluorescent microplate reader. Data are normalized to 2 min fluorescent intensity levels at 100%. Data are expressed as mean ± SEM (n = 4). *P < 0.05 vs non-Tg rabbits by t-test.

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  Fig. 2.. Plasma total cholesterol and HDL-C levels of rabbits after feeding a cholesterol diet for 16 weeks Plasma total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels of transgenic (Tg) and non-Tg rabbits fed with an adjusted cholesterol-rich diet for 16 weeks. Data are expressed as mean ± SEM (n = 8–12). P > 0.05 vs non-Tg rabbits by two-way repeated measure ANOVA.

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  Fig. 3.. Plasma lipoprotein profiles of rabbits on a chow diet and a cholesterol diet Plasma lipoproteins were separated by sequential gradient ultracentrifugation from plasma of rabbits on a normal (A) and an adjusted cholesterol diet for 8 weeks (B). Total cholesterol (TC), triglycerides (TG), and phospholipids (PL) in lipoproteins were measured. Data are expressed as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 vs non-Tg rabbits by t-test.

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  Supplementary Fig. 2.. Plasma lipoprotein and apolipoprotein analysis of rabbits on a cholesterol diet for 16 week Plasma lipoproteins were separated by sequential gradient ultracentrifugation. (A) Total cholesterol (TC), triglycerides (TG) and phospholipids (PL) in the lipoprotein fractions were measured. Data are expressed as mean ± SEM (n = 6). P > 0.05 vs non-Tg rabbits by t-test. (B) Apolipoprotein distribution in the lipoprotein fractions were analyzed by 4–20% SDS-PAGE stained with Coomassie Brilliant Blue. Representative gel images from each group are shown.

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  Fig. 4.. Analysis of gross aortic lesions Transgenic (Tg) and non-Tg rabbits were fed an adjusted cholesterol diet for 16 weeks, and then the aortic lesions were quantified. Representative pictures of aortas stained with Sudan IV are shown (left). The lesion area, defined by the sudanophilic area, was quantified (right). Each dot represents the lesion area of an individual animal (n = 8–12). P > 0.05 vs non-Tg rabbits by t-test.

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  Fig. 5.. Microscopic analysis of aortic atherosclerosis Representative micrographs of the aortic arch lesions from transgenic (Tg) and non-Tg rabbits are shown (top). Serial paraffin sections of the aortic arch were stained with hematoxylin-eosin (HE) and elastica van Gieson (EVG) or immunohistochemically stained with monoclonal antibodies against either macrophages or smooth muscle α-actin for smooth muscle cells (SMC). Intimal lesions on EVG stained sections and positively stained areas of macrophage and SMC were quantified (bottom). Data are expressed as mean ± SEM (n = 8–12). P > 0.05 vs non-Tg rabbits by t-test.

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  Fig. 6.. Analysis of coronary atherosclerosis Representative micrographs of the coronary lesions stained with hematoxylin-eosin from transgenic (Tg) and non-Tg rabbits are shown (left). The lesion size, expressed as stenosis % of coronary arteries, was quantified (right). Data are expressed as mean ± SEM (n = 7–11). P > 0.05 vs non-Tg rabbits by t-test.

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  Supplementary Fig. 3.. Analysis of lipoprotein lipase activity Post-heparinized plasma from non-Tg and Tg fed a cholesterol diet for 16 weeks were collected and lipoprotein lipase (LPL) activity was measured using a commercial colorimetric assay kit (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan). Data are expressed as mean ± SEM (n = 8–12).