G.S. Ganzetti

A salmon protein hydrolysate exerts lipid-independent anti-atherosclerotic activity in ApoE-deficient mice.

Fish consumption is considered health beneficial as it decreases cardiovascular disease (CVD)-risk through effects on plasma lipids and inflammation. We investigated a salmon protein hydrolysate (SPH) that is hypothesized to influence lipid metabolism and to have anti-atherosclerotic and anti-inflammatory properties. 24 female apolipoprotein (apo) E−/− mice were divided into two groups and fed a high-fat diet with or without 5% (w/w) SPH for 12 weeks. The atherosclerotic plaque area in aortic sinus and arch, plasma lipid profile, fatty acid composition, hepatic enzyme activities and gene expression were determined. A significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus was found in the 12 apoE−/− mice fed 5% SPH for 12 weeks compared to the 12 casein-fed control mice. Immunohistochemical characterization of atherosclerotic lesions in aortic sinus displayed no differences in plaque composition between mice fed SPH compared to controls. However, reduced mRNA level of Icam1 in the aortic arch was found. The plasma content of arachidonic acid (C20∶4n-6) and oleic acid (C18∶1n-9) were increased and decreased, respectively. SPH-feeding decreased the plasma concentration of IL-1β, IL-6, TNF-α and GM-CSF, whereas plasma cholesterol and triacylglycerols (TAG) were unchanged, accompanied by unchanged mitochondrial fatty acid oxidation and acyl-CoA:cholesterol acyltransferase (ACAT)-activity. These data show that a 5% (w/w) SPH diet reduces atherosclerosis in apoE−/− mice and attenuate risk factors related to atherosclerotic disorders by acting both at vascular and systemic levels, and not directly related to changes in plasma lipids or fatty acids.

An Immunomodulating Fatty Acid Analogue Targeting Mitochondria Exerts Anti-Atherosclerotic Effect beyond Plasma Cholesterol-Lowering Activity in apoE(-/-) Mice

Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator-activated receptors (PPARs) and proliferation of mitochondria. This study aimed to penetrate the effect of TTA on the development of atherosclerotic lesions in apolipoprotein (apo)-E-/- mice fed a high-fat diet containing 0.3% TTA for 12 weeks. These mice displayed a significantly less atherosclerotic development vs control. Plasma cholesterol was increased by TTA administration and triacylglycerol (TAG) levels in plasma and liver were decreased by TTA supplementation, the latter, probably due to increased mitochondrial fatty acid oxidation and reduced lipogenesis. TTA administration also changed the fatty acid composition in the heart, and the amount of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was reduced and increased, respectively. The heart mRNA expression of inducible nitric oxidase (NOS)-2 was decreased in TTA-treated mice, whereas the mRNA level of catalase was increased. Finally, reduced plasma levels of inflammatory mediators as IL-1α, IL-6, IL-17, TNF-α and IFN-γ were detected in TTA-treated mice. These data show that TTA reduces atherosclerosis in apoE-/- mice and modulates risk factors related to atherosclerotic disorders. TTA probably acts at both systemic and vascular levels in a manner independent of changes in plasma cholesterol, and triggers TAG catabolism through improved mitochondrial function.

Magnetic Resonance Imaging Visualization of Vulnerable Atherosclerotic Plaques at the Brachiocephalic Artery of Apolipoprotein E Knockout Mice by the Blood-pool Contrast Agent B22956/1

The aim of this study was to identify, by magnetic resonance imaging (MRI), the ability of the blood-pool contrast agent B22956/1 to detect atherosclerotic plaques developing at the brachiocephalic artery of apolipoprotein E knockout (apoE-KO) mice and to possibly identify vulnerable atherosclerotic lesions. After high-fat feeding for 8 or 12 weeks, MRIs of brachiocephalic arteries were acquired before and after B22956/1 administration; then vessels were removed and analyzed by histology. B22956/1 injection caused a rapid increase in plaque signal enhancement and plaque to muscle contrast values, which remained stable up to 70 minutes. A linear correlation between signal enhancement and macrophage content was found 10 minutes after B22956/1 injection (p < .01). Signal enhancement and plaque to muscle contrast values correlated with macrophage content 40 minutes after contrast agent administration (p < .01). Finally, 70 minutes after B22956/1 infusion, plaque to muscle contrast significantly correlated with the percentage of stenosis (p < .005). B22956/1 administration to high fat-fed apoE-KO mice resulted in a rapid enhancement of atherosclerotic plaques and in a great ability to rapidly visualize vulnerable plaques, characterized by a high macrophage content. These results suggest that B22956/1 could represent an interesting tool for the identification of atherosclerotic plaques potentially leading to acute cardiovascular events.The aim of this study was to identify, by magnetic resonance imaging (MRI), the ability of the blood-pool contrast agent B22956/1 to detect atherosclerotic plaques developing at the brachiocephalic artery of apolipoprotein E knockout (apoE-KO) mice and to possibly identify vulnerable atherosclerotic lesions. After high-fat feeding for 8 or 12 weeks, MRIs of brachiocephalic arteries were acquired before and after B22956/1 administration; then vessels were removed and analyzed by histology. B22956/1 injection caused a rapid increase in plaque signal enhancement and plaque to muscle contrast values, which remained stable up to 70 minutes. A linear correlation between signal enhancement and macrophage content was found 10 minutes after B22956/1 injection ( p < .01). Signal enhancement and plaque to muscle contrast values correlated with macrophage content 40 minutes after contrast agent administration ( p < .01). Finally, 70 minutes after B22956/1 infusion, plaque to muscle contrast significantly correlated with the percentage of stenosis ( p < .005). B22956/1 administration to high fat-fed apoE-KO mice resulted in a rapid enhancement of atherosclerotic plaques and in a great ability to rapidly visualize vulnerable plaques, characterized by a high macrophage content. These results suggest that B22956/1 could represent an interesting tool for the identification of atherosclerotic plaques potentially leading to acute cardiovascular events.

Liver-specific deletion of the Plpp3 gene alters plasma lipid composition and worsens atherosclerosis in apoE −/− mice

The PLPP3 gene encodes for a ubiquitous enzyme that dephosphorylates several lipid substrates. Genome-wide association studies identified PLPP3 as a gene that plays a role in coronary artery disease susceptibility. The aim of the study was to investigate the effect of Plpp3 deletion on atherosclerosis development in mice. Because the constitutive deletion of Plpp3 in mice is lethal, conditional Plpp3 hepatocyte-specific null mice were generated by crossing floxed Plpp3 mice with animals expressing Cre recombinase under control of the albumin promoter. The mice were crossed onto the athero-prone apoE−/− background to obtain Plpp3f/fapoE−/−Alb-Cre+ and Plpp3f/fapoE−/−Alb-Cre− offspring, the latter of which were used as controls. The mice were fed chow or a Western diet for 32 or 12 weeks, respectively. On the Western diet, Alb-Cre+ mice developed more atherosclerosis than Alb-Cre− mice, both at the aortic sinus and aorta. Lipidomic analysis showed that hepatic Plpp3 deletion significantly modified the levels of several plasma lipids involved in atherosclerosis, including lactosylceramides, lysophosphatidic acids, and lysophosphatidylinositols. In conclusion, Plpp3 ablation in mice worsened atherosclerosis development. Lipidomic analysis suggested that the hepatic Plpp3 deletion may promote atherosclerosis by increasing plasma levels of several low-abundant pro-atherogenic lipids, thus providing a molecular basis for the observed results.

Effect of Dietary Components from Antarctic Krill on Atherosclerosis in apoE-Deficient Mice

SCOPE Antarctic krill is a great source of n-3 fatty acids and high-quality proteins. Aim of the study was to evaluate the effect of Antarctic krill components on plasma lipids and atherosclerosis development. METHODS AND RESULTS Sixty apoEKO mice were divided into four groups and fed Western diet (CONTROL) or Western-like diets, differing for protein or fat content. Specifically, casein or fat in CONTROL was partially replaced by krill proteins (PRO), krill oil (KRILL OIL), or both (KRILL OIL+PRO). In KRILL OIL+PRO and KRILL OIL, cholesterol levels were significantly lower than in CONTROL group. Atherosclerosis in aorta of PRO, KRILL OIL and KRILL OIL+PRO was lower than in CONTROL, whereas, at the aortic sinus, atherosclerosis reduction was only observed in KRILL OIL. Liver steatosis, commonly present in CONTROL and PRO animals, was sporadic in KRILL OIL+PRO and KRILL OIL mice. Krill oil containing diets affected the expression of genes involved in cholesterol metabolism, mainly HMG-CoA reductase. No reduced systemic inflammation was found in all groups. CONCLUSION Krill oil containing diets were able to reduce cholesterol levels, inhibit plaque development and prevent liver damage. Krill proteins also reduced atherosclerosis development through mechanisms not involving lipid metabolism.

High-density lipoprotein deficiency in genetically modified mice deeply affects skin morphology: A structural and ultrastructural study.

Cutaneous lipids, endogenously synthetized and transported by lipoproteins, play a pivotal role in maintaining skin barrier. An impairment of extracutaneous lipid trafficking leads to the development of xanthomas, mostly arising in hyperlipidemic patients, but also in subjects with high-density lipoprotein (HDL) deficiency. The aim of this work was to evaluate, in a genetically modified mouse model, lacking two protein components of HDL particles, apolipoprotein(apo)E and apoA-I, the effect of HDL deficiency on skin morphology. Control mice (C57BL/6), apoE deficient mice (EKO), apoA-I deficient mice (A-IKO) and apoA-I/apoE double knockout mice (A-IKO/EKO) were maintained on a low-fat/low-cholesterol diet up to 30 weeks of age. At sacrifice, skin biopsies were processed for light (LM) and transmission electron microscopy (TEM). Whereas the skin of EKO, A-IKO, and C57BL/6 mice was comparable, LM analysis in A-IKO/EKO mice showed an increase in dermal thickness and the presence of foam cells and T lymphocytes in reticular dermis. TEM analysis revealed the accumulation of cholesterol clefts in the papillary dermis and of cholesterol crystals within foam cells. In conclusion, A-IKO/EKO mice represent an experimental model for investigating the cutaneous phenotype of human HDL deficiency, thus mimicking a condition in which human xanthomatous lesions can develop.