Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E-/- mice and rabbits fed a high-fat and -cholesterol diet

Abstract

Background: Glycosphingolipids, integral components of the cell membrane, have been shown to serve as messengers, transducing growth factor-initiated phenotypes. Here, we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and rabbits.

Methods and results: Apolipoprotein E(-/-) mice (12 weeks of age; n=6) were fed regular chow or a Western diet (1.25% cholesterol, 2% fat). Mice were fed 5 or 10 mg/kg of an inhibitor of glycosphingolipid synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), solubilized in vehicle (5% Tween-80 in PBS); the placebo group received vehicle only. At 20 and 36 weeks of age, serial echocardiography was performed to measure aortic intima-media thickening. Aortic pulse-wave velocity measured vascular stiffness. Feeding mice a Western diet markedly increased aortic pulse-wave velocity, intima-media thickening, oxidized low-density lipoprotein, Ca(2+) deposits, and glucosylceramide and lactosylceramide synthase activity. These were dose-dependently decreased by feeding D-PDMP. In liver, D-PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP2, low-density lipoprotein receptor, HMGCo-A reductase, and the cholesterol efflux genes (eg, ABCG5, ABCG8). D-PDMP affected very-low-density lipoprotein catabolism by increasing the gene expression for lipoprotein lipase and very-low-density lipoprotein receptor. Rabbits fed a Western diet for 90 days had extensive atherosclerosis accompanied by a 17.5-fold increase in total cholesterol levels and a 3-fold increase in lactosylceramide levels. This was completely prevented by feeding D-PDMP.

Conclusions: Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E(-/-) mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to ameliorate atherosclerosis and arterial stiffness.

Keywords: atherosclerosis; glycosphingolipids; mice, knockout; molecular imaging.

Figure 1

Aortic wall thickening and pulse wave velocity in atherosclerotic mice is ameliorated by treatment with D-PDMP. Aortic ultrasound imaging: 2DB-mode ultrasound images of the aorta from ApoE−/− mice fed mice chow. Top panel (A, B, C) shows 20 week old mice. Bottom panel (D, E, F) shows 36 week old mice. Control (A) ApoE−/− mice fed mice chow. ApoE−/− mouse fed high fat, high cholesterol (HFHC) diet plus vehicle (B), HFHC plus 10mpk of D-PDMP (C). Thirty six week old control ApoE−/− mice fed mice chow (D); placebo fed HFHC chow (E) and HFHC fed mice treated with 10mpk D-PDMP (F). Note the arrows indicate marked increase in aortic wall thickening in HFHC plus vehicle fed; placebo (B mice) mice at 20 weeks of age. This was followed by a marked increase in Ca²⁺ deposits at 36 weeks of age (marked by asterisk) in this group of mice as compared to control mice. This was prevented by feeding D-PDMP (F). Aortic wall thickness and aortic wall stiffness in atherosclerotic mice is ameliorated by treatment with D-PDMP shown in graphs (G) and (H). Quantitation of intima-media thickness (AoIMT) (G) and pulse wave velocity (PWV) respectively (H), in ApoE−/− mouse on a mice chow diet (control 0.63 ± 0.04 mm/4.37±0.26 m/s), HFHC diet plus vehicle (Placebo 1.21 ± 0.06 mm/6.38±0.89 m/s) (H), HFHC diet plus treatment with 5 and 10mpk of D-DPMP (1.04 ± 0.04mm/6.07±0.5 m/s and 0.73 ± 0.04 mm/4.24±0.15 m/s). Note that both AoIMT and PWV increase continuously in placebo mice from 12 weeks to 36 weeks and this was ameliorated upon treatment with D-PDMP in a dose-dependent manner. A two-way RM ANOVA using the Bonferroni multiplo comparisons post-test was performed..* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n = 3–5.

Figure 2

D-PDMP treatment ameliorates atherosclerotic plaque buildup and lumen volume in ApoE−/− mice fed a western diet. Masson Trichrome stained ascending aortic rings of ApoE−/− mouse: Control mice fed regular mice chow (A), mice fed high fat, high cholesterol (HFHC) diet consisting of 2% fat and 1.25 cholesterol plus vehicle (Placebo) (B), HFHC plus 5mpk, D-PDMP(C), and HFHC plus 10mpk, D-PDMP (D). Bar = 50 μm. Elastin fibers (E) decreased following treatment with D-PDMP. Quantification of lumen area (F) reveals a decrease in lumen volume in the mice fed HFHC. Lumen area is significantly reduced due to increased plaque accumulation in placebo mice aorta (Black arrow, B). Treatment with D-PDMP significantly reduced and/or delayed medial thickening, elastin fibers, and plaque accumulation fragmentation in a dose-dependent manner. A nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n = 3–5.

Figure 3

Glycosphingolipid glycosyltransferase activity in ApoE−/− mice fed high fat and high cholesterol diet with and without D-PDMP. Freshly harvested aorta tissue was stored in Tris HCl buffer supplemented with a protease inhibitor cocktail, homogenized and the activity of glucosylceramide synthase and lactosylceramide synthase were measured. Lactosylceramide synthase activity significantly decreased at 20 weeks (A) and 36 weeks (C) following treatment with D-PDMP. A significant change in glucosylceramide synthase activity was also observed at 20 weeks (B) and 36 weeks (D). In (E) and (F), treatment with D-PDMP decreases the mass of glycosphingolipids in the liver of ApoE−/− mice fed a western diet. Glycosphingolipids were extracted from 10 mg of harvested liver tissue and homogenized in CHCl3-MeOH, 2:1). Following treatment with the enzyme SCDase and derivatization by OPA, mass was calculated via liquid chromatography and tandem mass spectrometry against glucosylceramide and lactosylceramide standards. A significant decrease from the placebo in the mass of glucosylceramide was seen in the 5mpk treated and 10mpk treated mice fed the HFHC diet. A nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n = 3.

Figure 4

Plasma levels of oxidized LDL, cholesterol, triglycerides, and HDL-c in ApoE−/− mice fed a high fat and high cholesterol diet with and without D-PDMP. Serum extracted from ApoE−/− mice was analyzed for the presence of oxLDL (A), LDLc (B), triglycerides (C), and HDLc (D) on microtiter plates. Serum concentrations of oxLDL were determined using an immunohistochemical ELISA assay with an antibody against oxLDL. Concentrations of LDLc triglycerides and HDLc concentrations were taken from microtiter readings following Wako kit assays. Values are means ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; n = 3.

Figure 5

Effect of D-PDMP on the expression of hepatic genes, which play roles in cholesterol and lipid metabolism. Expression of hepatic genes involved in LDL metabolism; 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr), Low density lipoprotein receptor (Ldlr), Sterol regulatory element binding transcription factor 1(Srebf1), Sterol regulatory element binding transcription factor 2(Srebf2) (A) HDL metabolism; apolipoprotein A-I(Apoa1), scavenger receptor class B (SRB-1), CD36 antigen (B) VLDL and Triglyceride metabolism; lipoprotein lipase (Lpl), very low density lipoprotein receptor (Vldlr) (C) and Cholesterol efflux and uptake; ATP-binding cassette sub-family ABCA1(Abca1), cholesterol 7 alpha-hydroxylase(Cyp7A1) (D) as assessed by quantitative real-time PCR. Expressions of these genes were significantly up regulated in mice treated with D-PDMP in a dose dependent manner. Values are means ± SEM. A nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001; n = 3.

Figure 6

Western blot analysis of liver Srebp-2, LDLR, ABCG5 and ABCG8 proteins. Western blots illustrate D-PDMP induces the expression of Srebp-2 (A), LDL receptor (B), ABCG5 (C) and ABCG8 (D) proteins in liver tissues. Values are means ± SEM. A nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. * p ≤ 0.05, **** p ≤ 0.0001; n = 3.

Figure 6

Western blot analysis of liver Srebp-2, LDLR, ABCG5 and ABCG8 proteins. Western blots illustrate D-PDMP induces the expression of Srebp-2 (A), LDL receptor (B), ABCG5 (C) and ABCG8 (D) proteins in liver tissues. Values are means ± SEM. A nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. * p ≤ 0.05, **** p ≤ 0.0001; n = 3.

Figure 7

Inhibiting glycosphingolipid synthesis inhibits atherosclerosis in rabbits. New Zealand white rabbits (n=3) were fed a diet with and without cholesterol (2%) and fat (14%) and with and without D-PDMP (10mpk) for three months. Marked increase in lactosylceramide levels in the aorta tissue was observed (A) accompanied by extensive atherosclerosis and marked increase (17-fold) in serum cholesterol levels (B) were found in rabbits fed the hyperlipidemic diet in comparison to control and drug-treated rabbits at 3 months. This was prevented by treatment with D-PDMP. Statistical analysis: In 7A, a nonparametric one-way ANOVA using the Kruskal-Wallis test and Dunn’s multiple comparison post-test were performed. In 7B, a two-way repeated measures ANOVA was performed with Bonferroni’s multiple comparisons post-test. * p ≤ 0.05, **** p ≤ 0.0001; n = 3.

Figure 8

D-PDMP inhibits ox-LDL induced LCS activity and atherosclerosis development. Oxidized LDL (ox-LDL) activates LacCer synthase (LCS) to produce large quantities of LacCer, which stimulates the activity of NADPH oxidase. Concomitantly, there is an increase in the generation of superoxide radicals (ROS). ROS mediates p44MAPK phosphorylation thereby stimulating c-fos expression, promoting cell proliferation of vascular-smooth-muscle cells further to angiogenesis and generation of atherosclerosis. D-PDMP, a potent inhibitor of LCS, impaired ox-LDL mediated induction of LCS activity thereby inhibiting the above pathway.