Diabetic atherosclerosis in APOE*4 mice: synergy between lipoprotein metabolism and vascular inflammation

Abstract

Diabetes is a major risk factor for cardiovascular disease. To examine how diabetes interacts with a mildly compromised lipid metabolism, we introduced the diabetogenic Ins2(C96Y/+) (Akita) mutation into mice expressing human apoE4 (E4) combined with either an overexpressing human LDL receptor gene (hLDLR) or the wild-type mouse gene. The hLDLR allele caused 2-fold reductions in plasma HDL-cholesterol, plasma apoA1, and hepatic triglyceride secretion. Diabetes increased plasma total cholesterol 1.3-fold and increased apoB48 secretion 3-fold, while reducing triglyceride secretion 2-fold. Consequently, diabetic E4 mice with hLDLR secrete increased numbers of small, cholesterol-enriched, apoB48-containing VLDL, although they have near normal plasma cholesterol (<120 mg/dl). Small foam cell lesions were present in the aortic roots of all diabetic E4 mice with hLDLR that we analyzed at six months of age. None were present in nondiabetic mice or in diabetic mice without hLDLR. Aortic expression of genes affecting leukocyte recruitment and adhesion was enhanced by diabetes. ApoA1 levels, but not diabetes, were strongly correlated with the ability of plasma to efflux cholesterol from macrophages. We conclude that the diabetes-induced proinflammatory changes in the vasculature and the hLDLR-mediated cholesterol accumulation in macrophages synergistically trigger atherosclerosis in mice with human apoE4, although neither alone is sufficient.

Fig. 1.

Plasma glucose, insulin, and lipoproteins. A–D: Plasma glucose (A), insulin (B), triglycerides (C), and cholesterol (D) were measured following a 4 h fast in 4–5 month old nondiabetic and diabetic E4 mice without (white bars) and with hLDL (black bars). The numbers of mice used in each group are shown inside bars. *P < 0.05, **P < 0.001. E: Pooled plasma (100 µl) from nondiabetic E4 and E4h (left panel) and diabetic E4A and E4hA (right panel) mice was separated on a FPLC column, and fractions were analyzed for cholesterol (top) and triglycerides (bottom). Non-HDL to HDL ratio is shown in parentheses. Plasma was pooled from 6–8 mice of each genotype. F, G: ApoB48 (F), apoA1 (G), and apoE (H) in the plasma were measured by Western blot analyzed by densitometry and expressed relative to the mean of E4 plasma as 100. Representative bands from each genotype are shown. I: Apolipoprotein and lipid compositions of VLDL fraction isolated by ultracentrifugation. VLDL fractions from 175 μl plasma of E4 and E4h and from 50 μl plasma of E4A and E4hA were subjected to a SDS gel electrophoresis and stained with Coomassie Brilliant Blue. Pie charts on the right show lipid compositions of E4A-VLDL and E4hA-VLDL.

Fig. 2.

Hepatic lipids and VLDL secretion. A, B: Liver cholesterol (A) and triglycerides (B) in nondiabetic and diabetic E4 (white bars) and E4h mice (black bars). Numbers of mice used in each group are shown inside bars. *P < 0.05. C, D: Hepatic secretion of VLDL triglycerides (C) and cholesterol (D) following injection of the detergent Tyloxapol via the tail vein. Triangles are nondiabetic mice (left panel), and squares are diabetic mice (right panel). Open symbols are mice without hLDLR, and filled symbols are mice with hLDLR. F: ApoB was measured by Western blot in pooled samples (n = 5) of VLDL (d < 1.006 g/ml density fractions) at 2, 60, and 120 min postinjection.

Fig. 3.

Atherosclerotic lesions at the aortic root. At six months of age, mice were euthanized and perfused with 4% PFA, and 8 µm sections of the aortic root were cryosectioned. Sections were stained with Sudan IVB to highlight lipid (red) and counterstained with hematoxylin. Representative images of aortic root vessel walls are shown for E4h (A), E4A (B), and E4hA (C–G) mice. None of the examined mice from the E4, E4h, or E4A group but all from the E4hA group showed signs of fatty streaks or macrophage foam cell formation. Black arrows in (C) and (D) indicate fatty intimal depositions. White arrows in (E) and (F) indicate foam cell infiltration in the medial layers. G: Moma2 immunostaining of fatty intimal depositions (red on the bright field) in the neighboring section of (E).

Fig. 4.

Macrophage and vascular functions. A: Macrophage LDL uptake. Primary macrophages isolated from E4 mice (white bars) or E4h mice (black bars) were cultured in 5 mM (low) or 25 mM (high) glucose medium for 48 h. Macrophages were cultured with 1 µg/ml DiI-labeled human LDL (left side) or 1 µg/ml DiI-labeled oxidized human LDL (right side) for 4 h, and cellular florescence was measured to estimate LDL uptake. B: Cholesterol efflux to whole plasma correlates with plasma apoA1. Peritoneal macrophages from wild-type mice were labeled with [³H]cholesterol and incubated for 2.5 h with whole plasma (0.8%). Cholesterol efflux to individual plasma is expressed as proportion of labeled cholesterol moved from cells and plotted against their apoA1 levels estimated by Western blot. Open triangle, E4 mice; open circle, E4h mice; filled triangle, E4A mice; filled circle, E4hA mice. C: Cholesterol ester (CE), free cholesterol (FC), and triglyceride content of peritoneal macrophage. Statistics were with ANOVA, with hLDLR and diabetes as two factors.

Fig. 5.

Aortic gene expression of genes related to macrophage (A) and endothelial cells (B). Thoracic aortas were dissected from 4- to 5-month-old mice, and mRNA was isolated for gene expression analyses. Data are expressed as fold difference in log scale relative to the mean expression in E4 mice. The numbers of mice used are in parenthesis. Statistical significance of diabetic effects and hLDLR effects are by two-way ANOVA.

Fig. 6.

Alterations of vascular environment by diabetes and by hLDLR in mice expressing human apoE4. Arrows moving from left to right indicate the effects of an increase in LDLR expression as a result of the hLdlr allele. Arrows moving from top to bottom indicate the effects of diabetes due to Ins2^Akita/+. Nondiabetic E4 mice in top left carry most of plasma cholesterol in HDL (blue circles). Increased LDLR reduces HDL cholesterol in E4h (top right). Diabetes increases plasma glucose (black dots) and VLDL secretion (E4Akita, bottom left) and in E4hAkita mice (bottom right). Interactions between hLDLR and apoE4 compromise the clearance of remnants in E4hAkita mice, leading to an accumulation of VLDL remnants (red circles). In the vasculature, hLDLR increases VCAM-1 gene expression, whereas diabetes increases expression of ICAM-1, SELE, and MCP-1 (upward arrows). Additionally, hLDLR enhances cholesterol uptake while reduces efflux in apoE4-expressing macrophages, leading to foam cell formation only in E4hAkita mice.