Pleiotropic Effects of Atorvastatin Result in a Downregulation of the Carboxypeptidase U System (CPU, TAFIa, CPB2) in a Mouse Model of Advanced Atherosclerosis

Statins (hydroxymethyl-glutaryl-CoA-reductase inhibitors) lower procarboxypeptidase U (proCPU, TAFI, proCPB2). However, it is challenging to prove whether this is a lipid or non-lipid-related pleiotropic effect, since statin treatment decreases cholesterol levels in humans. In apolipoprotein E-deficient mice with a heterozygous mutation in the fibrillin-1 gene (ApoE−/−Fbn1C1039G+/−), a model of advanced atherosclerosis, statins do not lower cholesterol. Consequently, studying cholesterol-independent effects of statins can be achieved more straightforwardly in these mice. Female ApoE −/−Fbn1C1039G+/− mice were fed a Western diet (WD). At week 10 of WD, mice were divided into a WD group (receiving WD only) and a WD + atorvastatin group (receiving 10 mg/kg/day atorvastatin +WD) group. After 15 weeks, blood was collected from the retro-orbital plexus, and the mice were sacrificed. Total plasma cholesterol and C-reactive protein (CRP) were measured with commercially available kits. Plasma proCPU levels were determined with an activity-based assay. Total plasma cholesterol levels were not significantly different between both groups, while proCPU levels were significantly lower in the WD + atorvastatin group. Interestingly proCPU levels correlated with CRP and circulating monocytes. In conclusion, our results confirm that atorvastatin downregulates proCPU levels in ApoE−/−Fbn1C1039G+/− mice on a WD, and evidence was provided that this downregulation is a pleiotropic effect of atorvastatin treatment.

Candidate Gene Test for Quantitative Trait Loci Conferring Hypercholesterolemia on Mouse Chromosome 1

Objectives The TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a subcongenic mouse with TH donor segment, ∼25 Mb, on chromosome (Chr) 1 in a C57BL/6J (B6) background that harbors quantitative trait loci (QTL) conferring hypercholesterolemia, named Tchol1 (Tallyho Associated Cholesterol 1). The subcongenic mouse developed hypercholesterolemia compared to B6 mice demonstrating that distal segment of Chr 1 from TH genome is necessary to cause the hypercholesterolemia. In this study, we tested the candidacy of the apolipoprotein A2 (Apoa2) gene for Tachol1 by the quantitative complementation test. Apoa2, known regulator of cholesterol metabolism, maps to the Tchol1 locus. Methods To carry out the quantitative complementation test, both TH-homozygous Tachol1 subcongenic and B6-homozygous (B6) mice were mated to the Apoa2 knockout heterozygous [wild-type (wt)/null] mice to produce four types of animals; TH/wt, TH/null, B6/wt, and B6/null. Both male and female mice were weaned onto standard rodent chow and maintained. Blood was collected when animals were euthanized at 16 weeks of age. Total plasma cholesterol levels were determined using colorimetric assays. A two-way ANOVA was used to evaluate Apoa2 (null vs. wt) and Tachol1 (TH vs. B6) interaction effects for dependent variables, followed by the multiple comparison post test with Tukey correction using GraphPad Prism 8. Results Total plasma cholesterol levels were: 137 ± 5 (TH/wt), 119 ± 8 (TH/null), 103 ± 8 (B6/wt), and 80 ± 4 (B6/null) for males, and 149 ± 8 (TH/wt), 130 ± 9 (TH/null), 98 ± 3 (B6/wt), and 103 ± 6 (B6/null) for females [mean ± s.e.m; mg/dl]. Two-way ANOVA revealed no significant interaction between Tchol1 and Apoa2 knockout alleles for total plasma cholesterol levels in both males and females. However, there were significant main effects of Tchol1 and Apoa2 knockout alleles on total plasma cholesterol levels in males, while significant main effects of Tchol1 on them in females. Conclusions No significant interaction effect between knockout and QTL alleles is interpreted as evidence that the knockout locus is not equal to the QTL. Our results suggest that the Apoa2 gene is not identical to the Tchol1 QTL. Funding Sources AHA 18AIREA33960437, NIH 1 R15 DK113604-01A1, the WV-INBRE grant (P20GM103434), and the COBRE ACCORD grant (1P20GM121299).

Effects of salt reduction on cardiovascular risk factors. The STRIVE-study. A 4-month randomized controlled study among healthy families

Dietary salt intake in most countries is far beyond the recommended level and many countries have initiated national programs to decrease salt intake. However, potential adverse effects of reducing salt intake in the general population are currently discussed. Hence some randomized studies have shown adverse effects on plasma lipids, the renin-angiotensin-aldosterone system and the sympathetic nervous system, which may again increase risk of CVD. These findings are however mainly based on short-term studies with a very large acute salt reduction. This study aims to explore the effect of gradually reducing salt intake in a real life setting according to recommendations, and to examine the effect of different salt reduction strategies among families.The study was a 4-month cluster randomized controlled trial with families randomly assigned to either A) salt reduced bread, B) salt reduced bread and dietary counselling or C) standard bread (control) Participants in intervention A received bread gradually reduced in salt content from 1.2 g salt/100 g (regular) to 0.6 g salt/100 g in ryebread and 0.4 g salt/100 g in wheat bread. Participants in intervention B received the same salt reduced bread and in addition, they received dietary advise on how to further reduce their salt intake and promote potassium. The control group received regular bread. Changes in outcomes were assessed using linear mixed models.Results: A total of 89 families (155 adults; 156 children) from the suburb of Copenhagen, Denmark, participated in the study (February-July 2018). A total of 291 (94%) participants completed the intervention. Results presented are preliminary.Intention to treat analyses showed no significant effects of the salt reduction intervention on changes in systolic and diastolic blood pressure, plasma triglyceride. A small, but significant (-0.26 mmol/l; P = 0.02) decrease in total plasma cholesterol was shown in intervention group A. A similar, but non-significant, tendency was found in intervention group B. (more results will follow).Subgroup analyses, including only participants in the intervention groups that decreased their salt intake by at least 20% from baseline to 4-month follow-up, showed a significant decrease in diastolic (-3.5 mmHg; P < 0.0001) and systolic (-6.3 mmHg; P < 0.0001) blood pressure, total plasma cholesterol (-0.25 mmol/l; P = 0.0009), LDL plasma cholesterol (logarithm transformed) (-6%; P = 0.03) and plasma triglyceride (logarithm transformed) (-17%; P = 0.04). No significant effects were found for HDL plasma cholesterol, aldosterone, renin, plasma glucose and HbA1c.Conclusion: Reduced salt intake were associated with beneficial changes in cardiovascular risk factors. No adverse effects were observed.

Effects of Butter on Blood Biochemistry and Histotexure of Heart in the Development of Obesity in Swiss Albino Mice

The study was designed and performed in order to investigate the effects of butter on blood biochemistry and histo-texture of heart in mice. Thirty days old 30 Swiss Albino mice were divided into 3 equal groups (M, M1, and M2). Mice of group M were fed with normal mice pellet and were designated as control. Mice in the group M1 and M2 were fed with 10% and 20% butter supplemented feed respectively. In biochemical study, group M2 showed increase in total plasma cholesterol (P<0.001), LDL (P<0.001), TG (P<0.001), blood glucose (P<0.05), blood urea nitrogen (P<0.05) compared to mice in control group. In group of M1 there were also increase in total plasma cholesterol (P<0.01), LDL (P<0.001), TG (P<0.001), blood glucose (P<0.05), blood urea nitrogen (P<0.05) compared to mice in control group but lesser increased than the mice fed with 20% butter supplemented feed. In histopathological study there was no significant change in the heart of mice.DOI: http://dx.doi.org/10.3329/bjvm.v12i2.21293 Bangl. J. Vet. Med. (2014). 12 (2): 203-209

Abstract 20120: Protease-activated Receptor 4 Deficiency Reduces Atherosclerosis

Objective: Platelet activation is emerging as a critical process in both the formation and propagation of atherosclerosis. The coagulation protease thrombin activates platelets by cleavage of protease-activated receptor 4 (PAR4) in mice. Recent studies have demonstrated direct thrombin inhibitors reduce atherosclerosis in hypercholesterolemic mice. However, PAR4 deficiency had no effect on atherosclerosis in the apolipoprotein E (Apoe-/-) mouse model. The objective of this study was to re-examine the role of PAR4 deficiency in atherosclerosis using the alternative low-density lipoprotein receptor deficient mouse model. Methods and Results: To examine the effects of PAR4 deficiency on early atherosclerotic formation, low-density lipoprotein receptor deficient (Ldlr-/-) mice that were either PAR4+/+ (n = 11) or PAR4-/- (n = 12) were fed a fat-enriched diet (21% milk fat) and infused with angiotensin II (AngII; 1,000 ng/kg/min) for 28 days. PAR4 deficiency reduced AngII-induced aortic root atherosclerosis (+/+: 107 ± 22.1; -/-: 28.3 ± 7.50 μm2; P = 0.007). Bone marrow transplantation experiments were performed to determine the cellular source of PAR4 in atherosclerosis. Preliminary results demonstrate this reduction is dependent upon hematopoietic-derived PAR4 (+/+ into +/+: 230 ± 41; +/+ into -/-: 194 ± 23; -/- into +/+: 78 ± 18; and -/- into -/-: 68 ± 13 μm2; n = 4 each group; P < 0.02 +/+ hematopoietic versus -/- hematopoietic). PAR4 deficiency had no effects on total plasma cholesterol concentrations, lipoprotein-cholesterol distributions, or AngII increased systolic blood pressure. To determine the effects of a longer interval of diet-induced atherosclerosis, Ldlr-/-/PAR4+/+ (n = 12) or Ldlr-/-/PAR4-/- (n = 12) were fed a fat-enriched diet for 12 weeks. PAR4 deficiency attenuated aortic root atherosclerosis (+/+: 299 ± 15; -/-: 188 ± 18 μm2; P = 0.001) with no effects on total plasma cholesterol concentrations or lipoprotein distributions. Conclusion: We demonstrated that PAR4 deficiency resulted in an early (AngII: 74%) and late (Diet-induced: 37%) reduction in aortic root atherosclerosis. These results suggest that thrombin activation of PAR4 on platelets contributes to atherosclerosis in the Ldlr-/- model, but not the Apoe-/- model.

Abstract 207: Interesterified Fat Rich in Stearic Acid Increases Liver Neutrophil and M1 Macrophage Infiltration and Induces Nonalcoholic Steatohepatitis

Introduction: interesterified fats, rich in saturated fatty acid, have been used by food industry to replace trans. Palmitic acid is the most common dietary fat with known deleterious effects. On the hand stearic acid which is consumed in a low amount, has neutral effect on plasma lipids. Unlike vegetable oils, interesterified fats contain palmitic or stearic acid in the sn-2 position and the positional compositon of triglicerides may regulate its metabolic fate. Thus, the aim of this study was to evaluate the effect on the liver metabolism of the interesterified fats rich in stearic or palmitic fatty acids. Methods: Weaning LDLr-KO male mice were randomly distributed into five groups fed a high fat diet (40% of energy as fat) containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER) during 16 weeks. Total plasma cholesterol and triglyceride concentrations, liver total cholesterol, triglycerides and weight, neutrophil (neutr/area) and classically activated/inflammatory M1(CD11b) infiltrated macrophage were determined. Histological markers of NAFLD activity were assessed. Results: There were no differences in dietary intakes among all groups. The interesterification process did not alter plasma lipids concentrations (mg/dL±SD): PALM vs PALM INTER (499.2 ± 81.7 vs 526.8 ± 92.6) and STEAR vs STEAR INTER (342.6 ± 89.9 vs 363.9 ± 90.0). However, PALM and PALM INTER presented higher total plasma cholesterol, and STEAR and STEAR INTER showed total cholesterol concentrations similar to POLI (320.7±47.4). Liver weight, cholesterol and TG contents did not differ among the groups. POLI and STEARIC groups do not elicit NASH (score <3), PALM and PALM INTER presented intermediate stage (score ≤4) between STEARIC/POLI and STEARIC INTER. Nevertheless, STEAR INTER developed NASH (score ≥5), and induce higher neutrophil infiltration (5.53 ±4.1) as compared to all other groups. The M1 macrophage classically activated infiltration was also higher in STEAR INTER group than in all groups as demonstrated by imunofluorescence. Conclusion: STEAR INTER brings on NASH lesion, increased neutrophil and M1 macrophage classically activated/inflammatory infiltration in the liver.

Effects of oats on plasma cholesterol and lipoproteins in C57BL/6 mice are substrain specific

Cholesterol-lowering effects of oats have been demonstrated in both animals and human subjects. However, the crucial properties of oat-containing diets that determine their health effects need to be further investigated to optimise their use. A mouse model would be a valuable tool, but few such studies have been published to date. We investigated the effects of oat bran on plasma cholesterol and lipoproteins in two substrains of C57BL/6 mice. Western diet was made atherogenic by the addition of 0·8 % cholesterol and 0·1 % cholic acid. After 4 weeks on atherogenic diet, total plasma cholesterol had increased from 1·86–2·53 to 3·77–4·40 mmol/l. In C57BL/6NCrl mice, inclusion of 27 and 40 % oat bran reduced total plasma cholesterol by 19 and 24 %, respectively, reduced the shift from HDL to LDL+VLDL and caused increased faecal cholesterol excretion. There was no effect of oat bran on plasma levels of the inflammatory markers fibrinogen, serum amyloid A or TNF-α. Contrary to findings in C57BL/6NCrl mice, there was no sustained effect of oat bran (27 or 40 %) on plasma cholesterol in C57BL/6JBomTac mice after 4 weeks of feeding. Thus, C57BL/6NCrl mice fed an atherogenic diet are a good model for studies of physiological effects of oats, whereas a substrain derived from C57BL/6J, raised in a different breeding environment and likely possessing functional genetic differences from C57BL/6N, is considerably less responsive to oats. The present finding that two substrains of mice respond differently to oats is of practical value, but can also help to elucidate mechanisms of the cholesterol-lowering effect of oats.

Red algal cellular biomass lowers circulating cholesterol concentrations in Syrian golden hamsters consuming hypercholesterolaemic diets

Preliminary evidence suggests that consumption ofPorphyridium cruentum(PC) biomass results in hypocholesterolaemic effects; however, mechanisms responsible have not been elucidated. The aim of the present study was to determine whether PC biomass lowers circulating cholesterol concentrations, dose dependently, in hamsters fed hypercholesterolaemic diets for 28 d and determine whether cholesterol biosynthesis is affected. Biomass added to diets at 2·5, 5 and 10 % resulted in 14, 38 and 53 % reductions (P < 0·001) in total plasma cholesterol, respectively, compared with a control diet. Similarly, non-HDL-cholesterol concentrations in the 5 and 10 % PC groups were reduced (P < 0·001) 28 and 45 %, respectively,v.controls. These effects were unrelated to cholesterol fractional synthesis rate (FSR), as this did not differ between either treatment or control animals. PC consumption had no effect on food intake, plasma glucose concentrations or energy expenditure, but percentage of body fat was lower (P < 0·001) in the 5 and 10 % PC groups compared with controls. These data show that PC reduces total plasma cholesterol and non-HDL-cholesterol when incorporated into the diet at levels as low as 2·5 %. The mechanism of action for this reduction may be related to increased excretion since food intakes and cholesterol FSR were not reduced in the animals receiving the PC. In conclusion, the use of PC biomass reduces circulating cholesterol, dose dependently, in hypercholesterolaemic hamsters but not via reductions in cholesterol FSR. There is potential for the use of this biomass as a functional ingredient to aid in the management of blood cholesterol concentrations.