Wall Shear Stress (WSS) Analysis in Atherosclerosis in Partial Ligated Apolipoprotein E Knockout Mouse Model through Computational Fluid Dynamics (CFD).

Atherosclerosis involves an inflammatory response due to plaque formation within the arteries, which can lead to ischemic stroke and heart disease. It is one of the leading causes of death worldwide, with various contributing factors such as hyperlipidemia, hypertension, obesity, diabetes, and smoking. Wall shear stress (WSS) is also known as a contributing factor of the formation of atherosclerotic plaques. Since the causes of atherosclerosis cannot be attributed to a single factor, clearly understanding the mechanisms and causes of its occurrence is crucial for preventing the disease and developing effective treatment strategies. To better understand atherosclerosis and define the correlation between various contributing factors, computational fluid dynamics (CFD) analysis is primarily used. CFD simulates WSS, the frictional force caused by blood flow on the vessel wall with various hemodynamic changes. Using apolipoprotein E knockout (ApoE-KO) mice subjected to partial ligation and a high-fat diet at 1-week, 2-week, and 4-week intervals as an atherosclerosis model, CFD analysis was conducted along with the reconstruction of carotid artery blood flow via magnetic resonance imaging (MRI) and compared to the inflammatory factors and pathological staining. In this experiment, a comparative analysis of the effects of high WSS and low WSS was conducted by comparing the standard deviation of time-averaged wall shear stress (TAWSS) at each point within the vessel wall. As a novel approach, the standard deviation of TAWSS within the vessel was analyzed with the staining results and pathological features. Since the onset of atherosclerosis cannot be explained by a single factor, the aim was to find the correlation between the thickness of atherosclerotic plaques and inflammatory factors through standard deviation analysis. As a result, the gap between low WSS and high WSS widened as the interval between weeks in the atherosclerosis mouse model increased. This finding not only linked the occurrence of atherosclerosis to WSS differences but also provided a connection to the causes of vulnerable plaques.

Estradiol Protects Female ApoE KO Mice against Western-Diet-Induced Non-Alcoholic Steatohepatitis.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is higher in men than in women of reproductive age, and postmenopausal women are especially susceptible to developing the disease. AIM: we evaluated if female apolipoprotein E (ApoE) KO mice were protected against Western-diet (WD)-induced NASH. METHODS: Female ovariectomized (OVX) ApoE KO mice or sham-operated (SHAM) mice were fed either a WD or a regular chow (RC) for 7 weeks. Additionally, OVX mice fed a WD were treated with either estradiol (OVX + E2) or vehicle (OVX). RESULTS: Whole-body fat, plasma glucose, and plasma insulin were increased and associated with increased glucose intolerance in OVX mice fed a WD (OVX + WD). Plasma and hepatic triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) hepatic enzymes were also increased in the plasma of OVX + WD group, which was associated with hepatic fibrosis and inflammation. Estradiol replacement in OVX mice reduced body weight, body fat, glycemia, and plasma insulin associated with reduced glucose intolerance. Treatment also reduced hepatic triglycerides, ALT, AST, hepatic fibrosis, and inflammation in OVX mice. CONCLUSIONS: These data support the hypothesis that estradiol protects OVX ApoE KO mice from NASH and glucose intolerance.

Western diet feeding influences gut microbiota profiles in apoE knockout mice.

BACKGROUND: Gut microbiota plays an important role in many metabolic diseases such as diabetes and atherosclerosis. Apolipoprotein E (apoE) knock-out (KO) mice are frequently used for the study of hyperlipidemia and atherosclerosis. However, it is unknown whether apoE KO mice have altered gut microbiota when challenged with a Western diet. METHODS: In the current study, we assessed the gut microbiota profiling of apoE KO mice and compared with wild-type mice fed either a normal chow or Western diet for 12 weeks using 16S pyrosequencing. RESULTS: On a western diet, the gut microbiota diversity was significantly decreased in apoE KO mice compared with wild type (WT) mice. Firmicutes and Erysipelotrichaceae were significantly increased in WT mice but Erysipelotrichaceae was unchanged in apoE KO mice on a Western diet. The weighted UniFrac principal coordinate analysis exhibited clear separation between WT and apoE KO mice on the first vector (58.6%) with significant changes of two dominant phyla (Bacteroidetes and Firmicutes) and seven dominant families (Porphyromonadaceae, Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, Helicobacteraceae, Erysipelotrichaceae and Veillonellaceae). Lachnospiraceae was significantly enriched in apoE KO mice on a Western diet. In addition, Lachnospiraceae and Ruminococcaceae were positively correlated with relative atherosclerosis lesion size in apoE KO. CONCLUSIONS: Collectively, our study showed that there are marked changes in the gut microbiota of apoE KO mice, particularly challenged with a Western diet and these alterations may be possibly associated with atherosclerosis.

The effects of Zanthoxylum bungeanum extract on lipid metabolism induced by sterols.

Variant pharmacological activities of Zanthoxylum bungeanum were determined before. The aim of this study was to assess whether Z. bungeanum could regulate lipid metabolism. The cholesterol overloading HepG2 cells induced by sterols were used as in vitro model to study lipid-lowering activities of the n-butanol (BuOH) fraction isolated from Z. bungeanum (ZBBu). Male apolipoprotein E knockout (apoE-KO) mice with high fat diet were used as in vivo model. We firstly demonstrated ZBBu had effects on reversed lipid accumulation, decreased apoB and enhanced apoA1 secretion. It increased the amount of low density lipoprotein receptor (LDLR) protein, also significantly inhibited the expression of SREBP-1 and SREBP-2's target molecule (hydroxy methylglutaryl coenzyme A reductase, HMGCR), which might be active in stimulation of RCT. And the expression of genes involved in RCT, such as CYP27A1, LXR-α, ABCG1, was promoted by ZBBu. Furthermore, ZBBu could reduce serum TC, TG levels in apoE-KO mice. Our study indicated that ZBBu could regulate the lipid metabolism through increasing the amount of low density lipoprotein receptor (LDLR) and inducing the expression of genes involved in RCT.

Effects of Six Weeks of Hypoxia Exposure on Hepatic Fatty Acid Metabolism in ApoE Knockout Mice Fed a High-Fat Diet.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with a characteristic of abnormal lipid metabolism. In the present study, we employed apolipoprotein E knockout (ApoE KO) mice to investigate the effects of hypoxia exposure on hepatic fatty acid metabolism and to test whether a high-fat diet (HFD) would suppress the beneficial effect caused by hypoxia treatment. ApoE KO mice were fed a HFD for 12 weeks, and then were forwarded into a six-week experiment with four groups: HFD + normoxia, normal diet (ND) + normoxia, HFD + hypoxia exposure (HE), and ND + HE. The C57BL/6J wild type (WT) mice were fed a ND for 18 weeks as the baseline control. The hypoxia exposure was performed in daytime with normobaric hypoxia (11.2% oxygen, 1 h per time, three times per week). Body weight, food and energy intake, plasma lipid profiles, hepatic lipid contents, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and molecular/biochemical makers and regulators of the fatty acid synthesis and oxidation in the liver were measured at the end of interventions. Six weeks of hypoxia exposure decreased plasma triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) contents but did not change hepatic TG and non-esterified fatty acid (NEFA) levels in ApoE KO mice fed a HFD or ND. Furthermore, hypoxia exposure decreased the mRNA expression of Fasn, Scd1, and Srebp-1c significantly in the HFD + HE group compared with those in the HFD + normoxia group; after replacing a HFD with a ND, hypoxia treatment achieved more significant changes in the measured variables. In addition, the protein expression of HIF-1α was increased only in the ND + HE group but not in the HFD + HE group. Even though hypoxia exposure did not affect hepatic TG and NEFA levels, at the genetic level, the intervention had significant effects on hepatic metabolic indices of fatty acid synthesis, especially in the ND + HE group, while HFD suppressed the beneficial effect of hypoxia on hepatic lipid metabolism in male ApoE KO mice. The dietary intervention of shifting HFD to ND could be more effective in reducing hepatic lipid accumulation than hypoxia intervention.

High-Intensity Interval Training and Moderate-Intensity Continuous Training Attenuate Oxidative Damage and Promote Myokine Response in the Skeletal Muscle of ApoE KO Mice on High-Fat Diet.

The purpose of this study was to investigate the effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on the skeletal muscle in Apolipoprotein E knockout (ApoE KO) and wild-type (WT) C57BL/6J mice. ApoE KO mice fed with a high-fat diet were randomly allocated into: Control group without exercise (ApoE-/- CON), HIIT group (ApoE-/- HIIT), and MICT group (ApoE-/- MICT). Exercise endurance, blood lipid profile, muscle antioxidative capacity, and myokine production were measured after six weeks of interventions. ApoE-/- CON mice exhibited hyperlipidemia and increased oxidative stress, compared to the WT mice. HIIT and MICT reduced blood lipid levels, ROS production, and protein carbonyl content in the skeletal muscle, while it enhanced the GSH generation and potently promoted mRNA expression of genes involved in the production of irisin and BAIBA. Moreover, ApoE-/- HIIT mice had significantly lower plasma HDL-C content, mRNA expression of MyHC-IIx and Vegfa165 in EDL, and ROS level; but remarkably higher mRNA expression of Hadha in the skeletal muscle than those of ApoE-/- MICT mice. These results demonstrated that both exercise programs were effective for the ApoE KO mice by attenuating the oxidative damage and promoting the myokines response and production. In particular, HIIT was more beneficial to reduce the ROS level in the skeletal muscle.

Discovery, synthesis and anti-atherosclerotic activities of a novel selective sphingomyelin synthase 2 inhibitor.

The sphingomyelin synthase 2 (SMS2) is a potential target for pharmacological intervention in atherosclerosis. However, so far, few selective SMS2 inhibitors and their pharmacological activities were reported. In this study, a class of 2-benzyloxybenzamides were discovered as novel SMS2 inhibitors through scaffold hopping and structural optimization. Among them, Ly93 as one of the most potent inhibitors exhibited IC50 values of 91 nM and 133.9 µM against purified SMS2 and SMS1 respectively. The selectivity ratio of Ly93 was more than 1400-fold for purified SMS2 over SMS1. The in vitro studies indicated that Ly93 not only dose-dependently diminished apoB secretion from Huh7 cells, but also significantly reduced the SMS activity and increased cholesterol efflux from macrophages. Meanwhile, Ly93 inhibited the secretion of LPS-mediated pro-inflammatory cytokine and chemokine in macrophages. The pharmacokinetic profiles of Ly93 performed on C57BL/6J mice demonstrated that Ly93 was orally efficacious. As a potent selective SMS2 inhibitor, Ly93 significantly decreased the plasma SM levels of C57BL/6J mice. Furthermore, Ly93 was capable of dose-dependently attenuating the atherosclerotic lesions in the root and the entire aorta as well as macrophage content in lesions, in apolipoprotein E gene knockout mice treated with Ly93. In conclusion, we discovered a novel selective SMS2 inhibitor Ly93 and demonstrated its anti-atherosclerotic activities in vivo. The preliminary molecular mechanism-of-action studies revealed its function in lipid homeostasis and inflammation process, which indicated that the selective inhibition of SMS2 would be a promising treatment for atherosclerosis.

Procyanidins extracted from the litchi pericarp attenuate atherosclerosis and hyperlipidemia associated with consumption of a high fat diet in apolipoprotein-E knockout mice.

The beneficial effects of red wine against cardiovascular disease are associated with the abundant antioxidant polyphenols such as procyanidins. Recently, procyanidins extracted from the litchi pericarp (LPPC), a new source of procyanidins showed strong antioxidant activities in vitro, have been isolated and identified in our laboratory. The aim of present study was to investigate the anti-atherosclerotic effects of LPPC on atherosclerosis and hyperlipidemia in apolipoprotein E knockout (ApoE KO) mice fed a high fat diet (HFD, 21% fat, 0.15% cholesterol) for 24 weeks. The results showed that LPPC intervention alleviated atherosclerosis, fat accumulation and hyperlipidemia in ApoE KO mice. Furthermore, real-time RT-PCR results showed that LPPC can regulate several key genes involved in hepatic lipid homeostasis, such as increasing mRNA levels of farnesoid X receptor (FXR) and small heterodimer partner (SHP) which emerge as key regulators of lipid homeostasis at the transcriptional level, decreasing mRNA levels of 3-hydroxy-3-Methylglutaryl (HMG)-CoA reductase which mediates cholestrol biosynthesis, and up-regulating the mRNA expressions of ATP-binding cassette transporter-1 (ABCA1) which modulates cholesterol efflux. Thus, these results elucidated that LPPC could alleviate the lipid disorder especially hypercholesteromia and ameliorate atherosclerosis in ApoE-KO mice fed a WTD via regulating gene expression involved in hepatic lipid homeostasis effectively.

The Role of Apolipoprotein E and Ethanol Exposure in Age-Related Changes in Choline Acetyltransferase and Brain-Derived Neurotrophic Factor Expression in the Mouse Hippocampus.

Disruption of apolipoprotein E (APOE) is responsible for age-dependent neurodegeneration and cognitive impairment. Elderly individuals are more sensitive than young individuals to the effects of ethanol (EtOH), particularly those affecting cognition. We investigated the role of APOE deficiency and EtOH exposure on age-dependent alterations in choline acetyltransferase (ChAT) and brain-derived neurotrophic factor (BDNF) mRNA and protein expression in the mouse hippocampus. Three-month-old (young) and 12-month-old (aged) ApoE-knockout (ApoE-KO) and wild-type (WT) mice were treated with saline or 2 g/kg EtOH, and the bilateral hippocampus was collected after 60 min for real-time PCR and western blotting analyses. ChAT (P < 0.01) and BDNF (P < 0.01) expression were significantly decreased in both young and aged saline- and EtOH-treated ApoE-KO mice versus young and aged saline- and EtOH-treated WT mice. Aged saline- and EtOH-treated ApoE-KO mice exhibited greater differences in ChAT and BDNF expression (P < 0.01) than young saline- and EtOH-treated ApoE-KO mice. Aged EtOH-treated WT mice also exhibited larger decreases in BDNF expression (P < 0.01)-but not in ChAT expression-than young EtOH-treated WT mice. EtOH decreased ChAT and BDNF expression in both young (P < 0.01) and aged (P < 0.01) ApoE-KO mice versus EtOH-free ApoE-KO mice of the same age. EtOH also decreased BDNF expression in aged (P < 0.01) WT mice versus EtOH-free aged WT mice. In summary, these results suggest that APOE deficiency and EtOH exposure cause age-dependent decreases in ChAT and BDNF in the hippocampus. Importantly, the decreases in ChAT and BDNF were greater in aged EtOH-treated mice, particularly those lacking APOE, raising the possibility that APOE-deficient individuals who consume alcohol may be at greater risk of memory deficit.

Protective effect of betulinic acid on early atherosclerosis in diabetic apolipoprotein-E gene knockout mice.

Atherosclerosis, a chronic and progressive disease, is a leading cause of endothelial dysfunction, diabetes mellitus, hypertension, and hypercholesterolemia. Betulinic acid (BA), a pentacyclic triterpene, has been reported to have a variety of biological effects, including anti-inflammatory and immunomodulatory properties. This study was designed to determine whether BA could prevent atherosclerosis in diabetic apolipoprotein-E gene knockout (ApoE KO) mice. The mice were treated with BA for 12 weeks to examine its beneficial effects on atherosclerosis in ApoE KO mice. Male ApoE KO mice and age-matched control group mice (C57BL/6Jms) were used as experimental systems and their systolic blood pressure, insulin resistance, and vascular inflammation were measured. BA-treated ApoE KO mice showed lowered systolic blood pressure. The metabolic parameter showed that BA decreased blood urea nitrogen, triglyceride, and total cholesterol levels. Blood glucose, insulin, glucose tolerance results, and the homeostasis model assessment of insulin resistance (HOMA-IR) index were found to be better in BA-treated ApoE KO mice than untreated ApoE KO mice. Consistent with the change in lipid profiles, oil red O and H&E staining revealed that treatment with BA reduced atherosclerotic lesions such as roughened endothelial layers. BA ameliorated the reduction of endothelial nitric oxide synthase (eNOS) expression, leading to the inhibition of intracellular adhesion molecule 1 (ICAM-1) and endothelin 1 (ET-1) expression. These results suggest that BA may be useful in the treatment and prevention of early atherosclerosis via the attenuation of endothelial dysfunction in diabetic ApoE KO mice.

Magnetic Resonance Imaging of the Atherosclerotic Mouse Aorta.

Plaque development has been extensively studied using magnetic resonance imaging (MRI) in animal models of rapidly progressing atherosclerosis, such as apolipoprotein E-knockout (apoE-KO) mice. Preclinical MRI plays a significant role in the study of experimental atherosclerosis. Currently, MRI is capable of detecting luminal narrowing, plaque size, and morphology with high accuracy and reproducibility, providing reliable measurements of plaque burden. Therefore, MRI offers a noninvasive approach to serially monitor the progression of the disease. Compared with other imaging modalities, MRI appears to have the greatest potential for plaque characterization, through the use of multiple contrast weightings (e.g., T1, T2, and proton density). Here, we illustrate a standard procedure to image the aorta of atherosclerotic mice using noninvasive MRI.

Additive effects of lupin protein and phytic acid on aortic calcification in ApoE deficient mice.

Lupin proteins have repeatedly been shown to exhibit lipid lowering properties and reduce aortic calcification in atherosclerosis models. Despite many efforts on its identification, the component which is responsible for the observed effects is still under debate. Phytic acid which is generally associated with lupin protein isolates has currently been described as bioactive plant compound. The objective of the study was to determine the role of associated phytic acid for the described lupin protein effects. A two-factorial study with ApoE knockout mice was conducted in which mice received lupin protein isolate or casein with or without phytase. Phytic acid was added to the casein diets to a final concentration identical to the lupin protein diets. Here we show that the serum concentrations of cholesterol, lathosterol and desmosterol were lower and the faecal bile acid excretion was higher in the groups fed lupin proteins than in the groups fed casein (p < 0.05). Mice that received the lupin protein diet containing phytic acid were characterized by a lower aortic calcification than mice of the other three groups (p < 0.05). In conclusion, our results show that the cholesterol lowering properties of lupin protein isolate were not caused by phytic acid. However, the hypocalcific action of lupin proteins appears to depend on the combination of lupin proteins and phytic acid.