David C. Grimsditch

C3H apoE(: ) mice have less atherosclerosis than C57BL apoE(: ) mice despite having a more atherogenic serum lipid profile

Wild-type C57BL mice are known to be susceptible to diet-induced atherosclerosis, whilst C3H mice are resistant. We investigated the effect of these background strains on the hyperlipidaemia and atherosclerosis that develops in mice deficient in apolipoprotein E (apoE(-/-)). Male and female apoE(-/-) mice on C3H/HeNHsd (C3H) and C57BL/6J (C57) backgrounds were fed atherogenic Western diet for 12 weeks. Serum cholesterol and triglyceride concentrations were measured and atherosclerosis quantified in the aortic sinus. C3H apoE(-/-) mice fed normal diet had 1.5 2 fold higher serum cholesterol levels than C57 apoE(-/-) mice and 4-5 fold higher serum triglyceride concentrations. Feeding Western diet caused a 4-5 fold increase in serum cholesterol in all mice, but levels of triglyceride were either attenuated or were unaffected in C3H apoE(-/-) and C57 apoE(-/-) mice, respectively. C3H apoE(-/-) mice had approximately 2 fold higher serum cholesterol and 4 fold higher triglyceride concentrations than the C57 apoE(-/-) mice throughout the study. Serum triglyceride concentrations were 35-108% higher in male C3H apoE(-/-) than female C3H apoE(-/-) mice. Most of the lipids were present in the very low density lipoprotein (VLDL)/chylomicron fraction in both strains of mice whether they were fed normal or Western diet. Notwithstanding the lower plasma lipid concentrations, atherosclerotic lesion areas were more than 2-fold larger in C57 apoE(-/-) than in C3H apoE(-/-) mice (males 68 +/- 11 x 10(3) vs 30 +/- 6 x 10(3) females 102 +/- 12 x 10(3) vs 41 +/- 8 x 10(3) microm2. mean +/- SEM).

Metabolomic study of the LDL receptor null mouse fed a high-fat diet reveals profound perturbations in choline metabolism that are shared with ApoE null mice

Failure to express or expression of dysfunctional low-density lipoprotein receptors (LDLR) causes familial hypercholesterolemia in humans, a disease characterized by elevated blood cholesterol concentrations, xanthomas, and coronary heart disease, providing compelling evidence that high blood cholesterol concentrations cause atherosclerosis. In this study, we used (1)H nuclear magnetic resonance spectroscopy to examine the metabolic profiles of plasma and urine from the LDLR knockout mice. Consistent with previous studies, these mice developed hypercholesterolemia and atherosclerosis when fed a high-fat/cholesterol/cholate-containing diet. In addition, multivariate statistical analysis of the metabolomic data highlighted significant differences in tricarboxylic acid cycle and fatty acid metabolism, as a result of high-fat/cholesterol diet feeding. Our metabolomic study also demonstrates that the effect of high-fat/cholesterol/cholate diet, LDLR gene deficiency, and the diet-genotype interaction caused a significant perturbation in choline metabolism, notably the choline oxidation pathway. Specifically, the loss in the LDLR caused a marked reduction in the urinary excretion of betaine and dimethylglycine, especially when the mice are fed a high-fat/cholesterol/cholate diet. Furthermore, as we demonstrate that these metabolic changes are comparable with those detected in ApoE knockout mice fed the same high-fat/cholesterol/cholate diet they may be useful for monitoring the onset of atherosclerosis across animal models.

Dietary cholesterol reduces lipoprotein lipase activity in the atherosclerosis-susceptible Bio F 1 B hamster

We have compared lipoprotein metabolism in, and susceptibility to atherosclerosis of, two strains of male Golden Syrian hamster, the Bio F(1)B hybrid and the dominant spot normal inbred (DSNI) strain. When fed a normal low-fat diet containing approximately 40 g fat and 0.3 g cholesterol/kg, triacylglycerol-rich lipoprotein (chylomicron+VLDL) and HDL-cholesterol were significantly higher (P<0.001) in Bio F(1)B hamsters than DSNI hamsters. When this diet was supplemented with 150 g coconut oil and either 0.5 or 5.0 g cholesterol/kg, significant differences were seen in response. In particular, the high-cholesterol diet produced significantly greater increases in plasma cholesterol and triacylglycerol in the Bio F(1)B compared with the DSNI animals (P=0.002 and P<0.001 for cholesterol and triacylglycerol, respectively). This was particularly dramatic in non-fasting animals, suggesting an accumulation of chylomicrons. In a second experiment, animals were fed 150 g coconut oil/kg and 5.0 g cholesterol/kg for 6 and 12 months. Again, the Bio F(1)B animals showed dramatic increases in plasma cholesterol and triacylglycerol, and this was confirmed as primarily due to a rise in chylomicron concentration. Post-heparin lipoprotein lipase activity was significantly reduced (P<0.001) in the Bio F(1)B compared with the DSNI animals at 6 months, and virtually absent at 12 months. Bio F(1)B animals were also shown to develop significantly more (P<0.001) atherosclerosis. These results indicate that, in the Bio F(1)B hybrid hamster, cholesterol feeding reduces lipoprotein lipase activity, thereby causing the accumulation of chylomicrons that may be associated with their increased susceptibility to atherosclerosis.

Dietary Antioxidants Decrease Serum Soluble Adhesion Molecule (sVCAM-1, sICAM-1) but not Chemokine (JE/MCP-1, KC) Concentrations, and Reduce Atherosclerosis in C57BL but Not ApoE*3 Leiden Mice Fed an Atherogenic Diet

Dietary antioxidants are reported to suppress cellular expression of chemokines and adhesion molecules that recruit monocytes to the artery wall during atherosclerosis. In the present study we measured the effect of feeding apoE*3 Leiden mice or their non-transgenic (C57BL) littermates with atherogenic diets either deficient in, or supplemented with, dietary antioxidants (vitamin E, vitamin C and β-carotene) for 12 weeks, on serum levels of CC (JE/MCP-1) and CXC (KC) chemokines and soluble adhesion molecules (sVCAM-1, sICAM-1) and atherosclerotic lesion size. ApoE*3 Leiden mice developed gross hypercholesterolaemia, and markedly accelerated (10–20 fold; P < 0.0001) atherogenesis, compared with non-transgenic animals. Antioxidant consumption reduced lesion area in non-transgenic, but not apoE*3 Leiden, mice. Serum sVCAM-1 and sICAM-1 levels were significantly (P < 0.0001) increased (sVCAM-1 up to 3.9 fold; sICAM-1 up to 2.4 fold) by 4—8 weeks in all groups, and then declined. The initial increase in the concentration of adhesion molecules was reduced by 38%— 61% (P < 0.05) by antioxidant consumption, particularly in non-transgenic mice. By contrast, serum chemokine levels tended to increase more rapidly from baseline in apoE*3 Leiden mice, compared with non-transgenic animals, but were unaffected by dietary antioxidants. We conclude that dietary antioxidants reduce circulating soluble adhesion molecules and atherosclerosis in C57BL mice.

Temporal Relationships Between Circulating Levels of CC and CXC Chemokines and Developing Atherosclerosis in Apolipoprotein E*3 Leiden Mice

Objectives—CC and CXC chemokines are implicated in leukocyte recruitment during development of atherosclerotic lesions, suggesting circulating levels of chemokines may be useful serum markers of atherogenesis. Serum chemokine concentrations were measured in apolipoprotein (apo) E*3 Leiden mice and their nontransgenic littermates and related to the differing rates of atherogenesis in these animals. Methods and Results—Mice were fed a high-fat, high-cholesterol/cholate (HFC/C) diet for 18 weeks. Circulating levels of JE/monocyte chemotactic protein-1 increased (P <0.05) after 2 to 4 weeks, coincident with development of diet-induced hypercholesterolemia, and remained elevated throughout the study. Circulating KC concentrations increased (P <0.05) after consumption of HFC/C diet; however, unlike JE, serum KC concentrations increased more rapidly in apoE*3 Leiden mice than their nontransgenic littermates. Hepatic expression of JE and KC mRNA were detected by in situ hybridization in all mice fed HFC/C diet. Aortic expression of JE mRNA was seen only in apoE*3 Leiden mice within macrophage-rich atherosclerotic lesions. By contrast, no aortic expression of KC mRNA was detected by in situ hybridization. Conclusions—Increases in serum chemokine concentrations did not reflect temporal aortic production of these molecules and proved less predictive than serum cholesterol of the markedly different extent of atheroma in apoE*3 Leiden and nontransgenic mice.

Short term arterial remodelling in the aortae of cholesterol fed New Zealand white rabbits shown in vivo by high-resolution magnetic resonance imaging - implications for human pathology.

High-resolution, non-invasive imaging methods are required to monitor progression and regression of atherosclerotic plaques. We investigated the use of MRI to measure changes in plaque volume and vessel remodelling during progression and regression of atherosclerosis in New Zealand White rabbits. Atherosclerotic lesions were induced in the abdominal aorta by balloon injury and cholesterol feeding. MR images (2D) of the abdominal aorta were acquired with cardiac and respiratory gating using a fast spin echo sequence with and without fat-suppression. In an initial study on rabbits treated for 30 weeks we imaged the aortae with a spatial resolution of 250×250 micrometers with a slice thickness of 2 mm and achieved a close correlation between MRI-derived measurements and those made on perfusion pressure-fixed histological sections (r1 = 0.83, slope p1 < 0.01). We subsequently imaged 18 rabbits before and periodically during 12 weeks of cholesterol feeding (progression) followed by 12 weeks on normal diet (regression). Aortic wall (atherosclerotic lesion) volume increased significantly during progression and decreased during regression. In contrast, lumen volume increased during progression and did not change during regression. In conclusion, this study confirms that non-invasive, high-resolution MRI can be used to monitor progression and regression of atherosclerosis, each within 3 months and shows, for the first time in a short-term model, that positive remodelling occurs early during progression and persists through regression of atherosclerotic lesions.