Bacteroides fragilis Supplementation Deteriorated Metabolic Dysfunction, Inflammation, and Aorta Atherosclerosis by Inducing Gut Microbiota Dysbiosis in Animal Model.

BACKGROUND: The gut microbial ecosystem is an important factor that regulates host health and the onset of chronic diseases, such as inflammatory bowel diseases, obesity, hyperlipidemia, and diabetes mellitus, which are important risk factors for atherosclerosis. However, the links among diet, microbiota composition, and atherosclerotic progression are unclear. METHODS AND RESULTS: Four-week-old mice (-/- mice, C57Bl/6) were randomly divided into two groups, namely, supplementation with culture medium (control, CTR) and Bacteroides fragilis (BFS), and were fed a high-fat diet. The gut microbiota abundance in feces was evaluated using the 16S rDNA cloning library construction, sequencing, and bioinformatics analysis. The atherosclerotic lesion was estimated using Oil Red O staining. Levels of CD36, a scavenger receptor implicated in atherosclerosis, and F4/80, a macrophage marker in small intestine, were quantified by quantitative real-time PCR. Compared with the CTR group, the BFS group showed increased food intake, fasting blood glucose level, body weight, low-density lipoprotein level, and aortic atherosclerotic lesions. BFS dramatically reduced Lactobacillaceae (LAC) abundance and increased Desulfovibrionaceae (DSV) abundance. The mRNA expression levels of CD36 and F4/80 in small intestine and aorta tissue in the BFS group were significantly higher than those in the CTR group. CONCLUSIONS: gut microbiota dysbiosis was induced by BFS. It was characterized by reduced LAC and increased DSV abundance and led to the deterioration of glucose/lipid metabolic dysfunction and inflammatory response, which likely promoted aorta plaque formation and the progression of atherosclerosis.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression.

Background: Atherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD). Methods: miR-155 expression was quantified in aortae from ApoE-/- mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD. Results: Here, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs. Conclusion: miR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

Red Wine Grape Pomace Attenuates Atherosclerosis and Myocardial Damage and Increases Survival in Association with Improved Plasma Antioxidant Activity in a Murine Model of Lethal Ischemic Heart Disease.

A healthy dietary pattern and high quality nutrient intake reduce atherosclerotic cardiovascular disease risk. Red wine grape pomace (RWGP)-a rich natural source of dietary fiber and antioxidants-appears to be a potential functional food ingredient. The impact of a dietary supplementation with RWGP flour was evaluated in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice, a model of lethal ischemic heart disease. SR-B1 KO/ApoER61h/h mice were fed with atherogenic (high fat, cholesterol, and cholic acid, HFC) diet supplemented with: (a) 20% chow (HFC-Control), (b) 20% RWGP flour (HFC-RWGP), or (c) 10% chow/10% oat fiber (HFC-Fiber); and survival time was evaluated. In addition, SR-B1 KO/ApoER61h/h mice were fed for 7 or 14 days with HFC-Control or HFC-RWGP diets and plasma lipid levels, inflammation, oxidative damage, and antioxidant activity were measured. Atherosclerosis and myocardial damage were assessed by histology and magnetic resonance imaging, respectively. Supplementation with RWGP reduced premature death, changed TNF-α and IL-10 levels, and increased plasma antioxidant activity. Moreover, decreased atheromatous aortic and brachiocephalic plaque sizes and attenuated myocardial infarction and dysfunction were also observed. These results suggest that RWGP flour intake may be used as a non-pharmacological therapeutic approach, contributing to decreased progression of atherosclerosis, reduced coronary heart disease, and improved cardiovascular outcomes.

Danhong Injection Attenuates High-Fat-Induced Atherosclerosis and Macrophage Lipid Accumulation by Regulating the PI3K/AKT Insulin Pathway.

BACKGROUND AND AIMS: High-fat diet (HFD) is reported to induce atherosclerosis and insulin resistance. Macrophage lipid accumulation has been implicated as key mediators during the development of HFD-induced atherosclerosis. Traditional Chinese formula, which has long been used to improve disorder of glucose and lipid metabolism of patients, is now gradually being used as complementary therapy. This study aimed to investigate the effect of Danhong injection (DHI), a Chinese medicine used for the treatment of coronary artery disease, on atherosclerosis and its underlying mechanisms. METHODS AND RESULTS: We observed the effects of DHI on HFD-induced atherosclerosis in a mice model, macrophage lipid accumulation in an ox-LDL-stimulated macrophage model, and the role of PI3K/AKT insulin pathway in the process of DHI ameliorating atherosclerosis. The data demonstrated that DHI attenuated atherosclerosis by ameliorating blood lipids, reducing the atherosclerotic index and atherosclerotic plaque area in HFD-induced atherosclerotic mice, and inhibiting TC levels in an ox-LDL-induced macrophage model. By estimating the levels of serum insulin resistance-related indexes and protein expression of GLUT-4, DHI treatment dramatically inhibited the levels of fasting serum NEFA and fasting serum insulin and promoted the protein expression of GLUT-4 in aortas of the HFD-induced atherosclerotic mice. Moreover, according to the hints provided by microarray-based transcriptional profiling, the results demonstrated that DHI treatment also promoted the activation of PI3K/AKT insulin signaling pathway induced by IRS-1 in aortas of HFD-induced atherosclerotic mice. Furthermore, in an ox-LDL-induced macrophage model, the activation of PI3k/AKT signaling pathway also effectively functioned in the process of DHI inhibiting macrophage lipid accumulation. CONCLUSIONS: These results highlight that DHI treatment attenuates atherosclerosis and macrophage lipid accumulation by promoting the activation of PI3K/AKT insulin signaling pathway. It provides new insights into the molecular mechanism of DHI and its therapeutic potential in the treatment of atherosclerosis.

The promotion effect of novel magnetic nanoparticles on atherosclerotic plaque vulnerability in apolipoprotein E-/- mice.

Although manufactured magnetic nanoparticles (NPs) are currently used in many fields, NPs have potential toxicity on cardiovascular system especially atherosclerosis. In our previous study, we prepared novel Fe3O4 nanoparticles surface-coated with aminoguanidine (Fe3O4-AG NPs) which could remove acid dyes from aqueous solution efficiently. To understand its biocompatibility to atherosclerotic plaque vulnerability, we investigated the effects of the nanoparticles on human umbilical vein endothelial cells (HUVECs) in vitro and plaque stability in vivo. Fe3O4-AG NPs were taken up by HUVECs and induced HUVEC apoptosis. Fe3O4-AG NP injection remarkably promoted plaque vulnerability at low-dose (0.5 mg/kg) but not high-dose (5.0 mg/kg) in apolipoprotein E-/- (ApoE-/-) mice. Further study indicated that Fe3O4-AG NP-induced atherosclerotic plaque vulnerability was tightly linked to bioactivity of nitric oxide (NO). A significant decrease in NO production was induced which coincided with the inhibition of endothelial nitric oxide synthase (eNOS) activity in serum and endothelium of plaque in ApoE-/- mice injected with low-dose Fe3O4-AG NPs in vivo and HUVECs treated with low-dose Fe3O4-AG NPs in vitro. Thus, the low concentration of Fe3O4-AG NPs presented toxicity to atherosclerosis. Our results indicated that the use of Fe3O4-AG NPs to improve aqueous solution pollution should be cautious due to the potential toxicity.

LongShengZhi Capsule Reduces Established Atherosclerotic Lesions in apoE-Deficient Mice by Ameliorating Hepatic Lipid Metabolism and Inhibiting Inflammation.

Disorders of lipid metabolism and inflammation play an important role in atherosclerosis. LongShengZhi (LSZ) capsule, a Chinese herbal medicine, has been used for treatment of patients with vascular diseases for many years. In this article, we determined the effect of LSZ on the progression of established atherosclerotic lesions in apoE-deficient (apoE) mice. ApoE mice were prefed high-fat diet (HFD) for 8 weeks to induce atherosclerosis, then started with LSZ treatment contained in HFD for 10 weeks. Although LSZ had little effect on HFD-induced hypercholesterolemia, it substantially reduced en face and sinus aortic lesions. The reduction of lesions was associated with reduced macrophage/foam cell accumulation by activating ABCA1/ABCG1 expression. LSZ maintained the integrity of arterial wall by increasing collagen or smooth muscle cell content and inhibiting cell apoptosis. LSZ also attenuated HFD-induced fatty liver by down-regulating expression of lipogenic and cholesterol synthetic genes while activating expression of triglyceride catabolism genes. Moreover, LSZ demonstrated potent anti-inflammatory effects. In vivo, LSZ reduced serum TNF-α levels, infiltration of neutrophils, Kupffer cells, and expression of inflammatory cytokines in the liver. In vitro, it inhibited lipopolysaccharide or palmitate-induced expression of inflammatory cytokines in macrophages. Therefore, LSZ reduces atherosclerosis by ameliorating hepatic lipid metabolism and inhibiting inflammation.

The Inhibitory Effect of Ojeoksan on Early and Advanced Atherosclerosis.

Atherosclerosis is closely related to vascular dysfunction and hypertension. Ojeoksan (OJS), originally recorded in an ancient Korean medicinal book named "Donguibogam", is a well-known, blended herbal formula. This study was carried out to investigate the beneficial effects of OJS on atherosclerosis in vitro and in vivo. Western-diet-fed apolipoprotein-E gene-deficient mice (ApoE -/-) were used for this study for 16 weeks, and their vascular dysfunction and inflammation were analyzed. OJS-treated ApoE -/- mice showed lowered blood pressure and glucose levels. The levels of metabolic parameters with hyperlipidemia attenuated following OJS administration. Hematoxylin and eosin (H&E) staining revealed that treatment with OJS reduced atherosclerotic lesions. OJS also suppressed the expression of adhesion molecules and matrix metalloproteinases (MMPs) compared to Western-diet-fed ApoE -/- mice and tumor necrosis factor-alpha (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). Expression levels of MicroRNAs (miRNA)-10a, -126 3p were increased in OJS-fed ApoE -/- mice. OJS significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt), which are involved in nitric oxide (NO) production. OJS also regulated eNOS coupling by increasing the expression of endothelial GTP Cyclohydrolase-1 (GTPCH). Taken together, OJS has a protective effect on vascular inflammation via eNOS coupling-mediated NO production and might be a potential therapeutic agent for both early and advanced atherosclerosis.

A diet enriched with tree nuts reduces severity of atherosclerosis but not abdominal aneurysm in angiotensin II-infused apolipoprotein E deficient mice.

BACKGROUND AND AIMS: Diets enriched with tree nuts have been demonstrated to reduce the risk of atherosclerosis-related cardiovascular events. Abdominal aortic aneurysm (AAA) shares common risk factors with atherosclerosis and AAA patients commonly have atherosclerosis related cardiovascular events. AAA has some distinct pathological and clinical characteristics to those of atherosclerosis. No previous study has examined the effect of a diet enriched with tree nuts on experimental or clinical AAA. This study investigated the effect of a diet enriched with tree nuts on the development and severity of AAA within an experimental rodent model. METHODS: Male apolipoprotein E deficient mice were allocated to a diet enriched with tree nuts or control diet for 56 days (n = 17 per group). After 28 days, all mice were infused with angiotensin II whilst being maintained on their respective diets. The primary outcome was AAA severity assessed by the supra-renal aortic diameter, measured by ultrasound and ex vivo morphometric analysis. The severity of atherosclerosis was assessed by computer-aided analysis of Sudan IV stained aortic arches and sections of brachiocephalic arteries prepared with Van Gieson's stain. RESULTS: The diet enriched with tree nuts did not influence aortic diameter or aortic rupture incidence. Mice receiving the diet enriched with tree nuts had significantly less atherosclerosis within the brachiocephalic artery (p = 0.033) but not in the aortic arch. CONCLUSIONS: This experimental study suggests that a diet enriched with tree nuts does not reduce the severity of AAA, but does reduce the severity of atherosclerosis within the brachiocephalic artery. The study was not powered to identify a moderate effect of the diet on the primary outcome and therefore this cannot be excluded.

Impact of Gut Microbiota and Diet on the Development of Atherosclerosis in Apoe-/- Mice.

Objective- To investigate the effect of gut microbiota and diet on atherogenesis. Approach and Results- Here, we investigated the interaction between the gut microbiota and diet on atherosclerosis by feeding germ-free or conventionally raised Apoe-/- mice chow or Western diet alone or supplemented with choline (which is metabolized by the gut microbiota and host enzymes to trimethylamine N-oxide) for 12 weeks. We observed smaller aortic lesions and lower plasma cholesterol levels in conventionally raised mice compared with germ-free mice on a chow diet; these differences were not observed in mice on a Western diet. Choline supplementation increased plasma trimethylamine N-oxide levels in conventionally raised mice but not in germ-free mice. However, this treatment did not affect the size of aortic lesions or plasma cholesterol levels. Gut microbiota composition was analyzed by sequencing of 16S rRNA genes. As expected, the global community structure and relative abundance of many taxa differed between mice fed chow or a Western diet. Choline supplementation had minor effects on the community structure although the relative abundance of some taxa belonging to Clostridiales was altered. Conclusions- In conclusion, the impact of the gut microbiota on atherosclerosis is dietary dependent and is associated with plasma cholesterol levels. Furthermore, the microbiota was required for trimethylamine N-oxide production from dietary choline, but this process could not be linked to increased atherosclerosis in this model.

Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

BACKGROUND: CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. METHODS: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. RESULTS: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. CONCLUSIONS: These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in Apoe-/- mice.

BACKGROUND AND AIMS: Gut microbiota plays a major role in metabolic disorders. Berberine is used to treat obesity, diabetes and atherosclerosis. The mechanism underlying the role of berberine in modulating metabolic disorders is not fully clear because berberine has poor oral bioavailability. Thus, we evaluated whether the antiatherosclerotic effect of berberine is related to alterations in gut microbial structure and if so, whether specific bacterial taxa contribute to the beneficial effects of berberine. METHODS: Apoe-/- mice were fed either a normal-chow diet or a high-fat diet (HFD). Berberine was administered to mice in drinking water (0.5 g/L) for 14 weeks. Gut microbiota profiles were established by high throughput sequencing of the V3-V4 region of the bacterial 16S ribosomal RNA gene. The effects of berberine on metabolic endotoxemia, tissue inflammation and gut barrier integrity were also investigated. RESULTS: Berberine treatment significantly reduced atherosclerosis in HFD-fed mice. Akkermansia spp. abundance was markedly increased in HFD-fed mice treated with berberine. Moreover, berberine decreased HFD-induced metabolic endotoxemia and lowered arterial and intestinal expression of proinflammatory cytokines and chemokines. Berberine treatment increased intestinal expression of tight junction proteins and the thickness of the colonic mucus layer, which are related to restoration of gut barrier integrity in HFD-fed mice. CONCLUSIONS: Modulation of gut microbiota, specifically an increase in the abundance of Akkermansia, may contribute to the antiatherosclerotic and metabolic protective effects of berberine, which is poorly absorbed orally. Our findings therefore support the therapeutic value of gut microbiota manipulation in treating atherosclerosis.

Crocus sativus L. aqueous extract reduces atherogenesis, increases atherosclerotic plaque stability and improves glucose control in diabetic atherosclerotic animals.

BACKGROUND AND AIMS: We aimed to evaluate a possible atheroprotective effect of saffron aqueous extract (SFE), and its potential anti-inflammatory mechanisms, in apoE knockout (ApoE-/-) mice. METHODS: Fifty male, ApoE-/- mice, fed a high-fat diet (HFD) for 12 weeks, were randomized into 5 groups: (1) baseline group, euthanatized, without intervention, (2) three saffron groups, receiving HFD and 30,60,90 mg/kg/day of SFE, respectively, for four weeks, per os through gavage, after reconstitution in water for injection (WFI), (3) control group (COG), receiving daily HFD and the same volume of WFI (four weeks). After blood sampling and euthanasia, aortic roots were excised and analyzed for gene expression and/or percentage of aortic stenosis, relative content of macrophages, smooth muscle cells (SMCs), connective tissue, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases-2,-3,-9 (MMP-2,-3,-9) and their inhibitor (TIMP-2) and IL-6. SFE doses were determined by a pilot serum pharmacokinetic study in C57BL/6J wild-type mice. RESULTS: SFE did not affect body weight and total cholesterol levels (p > 0.05), while high SFE dose significantly ameliorated glucose and triglycerides profiles compared to other groups (p < 0.05). SFE considerably decreased aortic stenosis in a dose-dependent manner (p < 0.05). Furthermore, increasing SFE doses proportionally reduced macrophages content and increased within plaques content of collagen, elastin, and SMCs, promoting more stable plaque phenotype compared to COG (p < 0.05). Those effects seemed to be associated with a considerable reduction (>30%) in IL-6, TNF-α, MCP-1, MMP-2,-3,-9 (p < 0.05) and MMP-2/TIMP-2 ratio. CONCLUSIONS: SFE exerted dose-dependent anti-atherosclerotic and plaque-stabilizing effects in Apo-E-/- mice, probably mediated by a favorable modification of inflammatory mechanisms, which requires further investigation.

DBZ (Danshensu Bingpian Zhi), a Novel Natural Compound Derivative, Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice.

BACKGROUND: DBZ (Danshensu Bingpian Zhi), a synthetic derivative of a natural compound found in traditional Chinese medicine, has been reported to suppress lipopolysaccharide-induced macrophage activation and lipid accumulation in vitro. The aim of this study was to assess whether DBZ could attenuate atherosclerosis at early and advanced stages. METHODS AND RESULTS: The effects of DBZ on the development of atherosclerosis were studied using apolipoprotein E-deficient (apoE-/-) mice. For early treatment, 5-week-old apoE-/- mice were fed a Western diet and treated daily by oral gavage with or without DBZ or atorvastatin for 10 weeks. For advanced treatment, 5-week-old apoE-/- mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then they were randomly divided into 4 groups and subjected to the treatment of vehicle, 20 mg/kg per day DBZ, 40 mg/kg per day DBZ, or 10 mg/kg per day atorvastatin for the subsequent 10 weeks. We showed that early treatment of apoE-/- mice with DBZ markedly reduced atherosclerotic lesion formation by inhibiting inflammation and decreasing macrophage infiltration into the vessel wall. Treatment with DBZ also attenuated the progression of preestablished diet-induced atherosclerotic plaques in apoE-/- mice. In addition, we showed that DBZ may affect LXR (liver X receptor) function and that treatment of macrophages with DBZ suppressed lipopolysaccharide-stimulated cell migration and oxidized low-density lipoprotein-induced foam cell formation. CONCLUSIONS: DBZ potentially has antiatherosclerotic effects that involve the inhibition of inflammation, macrophage migration, leukocyte adhesion, and foam cell formation. These results suggest that DBZ may be used as a therapeutic agent for the prevention and treatment of atherosclerosis.

The effect of tocopheryl phosphates (TPM) on the development of atherosclerosis in apolipoprotein-E deficient mice.

α-Tocopheryl phosphate (TP) is a naturally occurring form of vitamin E found in the body. In the present study we compared the ability of an α-TP mixture (TPM) against a standard vitamin E supplement, α-tocopherol acetate (TA) on the development of atherosclerotic lesions in ApoE-deficient mice. Mice were maintained on either a normal chow diet for 24 weeks (Normal Diet), vs a group in which the final 8 weeks of the 24-week period mice were placed on a high fat (21%), high cholesterol (0.15%) challenge diet (HFHC), to exacerbate atherosclerotic lesion development.. The difference in these two control groups established the extent of the diet-induced atherosclerotic lesion development. Mice in the various treatment groups received either TA (300 mg/kg chow) or TPM (6.7-200 mg/kg chow) for 24 weeks, with TPM treatment resulting in dose-dependent significant reductions in atherosclerotic lesion formation and plasma levels of pro-inflammatory cytokines. TA-treated mice, with the tocopherol equivalent TPM dose (200 mg/kg chow), showed no significant reduction in plasma lipid levels or evidence for aortic lesion regression. At this TPM equivalent TA dose, a 44% reduction in aortic lesion formation was observed. In addition, these TPM treated mice, also showed a marked reduction in aortic superoxide formation and decreased circulating plasma levels of known pro-inflammatory markers IL-6, MCP-1, IL-1ß, IFN-γ and TNF-α. These findings indicate that TPM treatment slows progression of atherosclerotic lesions in ApoE-deficient mice with this effect potentially involving reduced oxidative stress and decreased inflammation.

Inhibitory effect of trans-caryophyllene (TC) on leukocyte-endothelial attachment.

trans-Caryophyllene (TC) is a major component found in the essential oils of many spices and foods/medicinal plants. It is a natural sesquiterpene and has been the subject of numerous studies. However, the effects of TC on vascular inflammation remain unknown. In this study, we reported that TC treatment in human umbilical vein endothelial cells (HUVECs) prevented attachment of monocytic leukemia cell line THP-1 cells to endothelial cells. In addition, in vivo results indicate that TC inhibited macrophage infiltration to the aortic surface and reduced total serum levels of cholesterol and triglycerides. Importantly, administration of TC could inhibit the induction of vascular cell adhesion molecule-1 (VCAM-1) both in vitro and in vivo. Notably, our data indicate that the inhibitory effects of TC on the expression of VCAM-1 are mediated by the JAK2/STAT1/IRF-1 pathway. TC is a specific agonist of the type 2 cannabinoid receptor (CB2R). Importantly, we further verified that the inhibitory effects of TC on the expression of IRF-1 and VCAM-1 are dependent on activation of CB2R. Inhibition of CB2R by either specific inhibitors or RNA interference abolished the inhibitory effects of TC on the expression of IRF-1 and VCAM-1. Our results suggest that TC might have a capacity to suppress the development of atherosclerosis.

Dietary supplementation with long-chain monounsaturated fatty acid isomers decreases atherosclerosis and alters lipoprotein proteomes in LDLr-/- mice.

BACKGROUND AND AIMS: Concentrated fish oils, containing a mixture of long-chain monounsaturated fatty acids (LCMUFA) with aliphatic chains longer than 18 C atoms (i.e., C20:1 and C22:1), have been shown to attenuate atherosclerosis development in mouse models. It is not clear, however, how individual LCMUFA isomers may act on atherosclerosis. METHODS: In the present study, we used saury fish oil-derived concentrates enriched in either C20:1 or C22:1 isomer fractions to investigate their individual effect on atherosclerosis and lipoprotein metabolism. LDLR-deficient (LDLr-/-) mice were fed a Western diet supplemented with 5% (w/w) of either C20:1 or C22:1 concentrate for 12 wk. RESULTS: Compared to the control Western diet with no supplement, both LCMUFA isomers increased hepatic levels of LCMUFA by 2∼3-fold (p < 0.05), and decreased atherosclerotic lesion areas by more than 40% (p < 0.05), although there were no major differences in plasma lipoproteins or hepatic lipid content. Both LCMUFA isomers significantly decreased plasma CRP levels, improved Abca1-dependent cholesterol efflux capacity of apoB-depleted plasma, and enhanced Ppar transcriptional activities in HepG2 cells. LC-MS/MS proteomic analysis of lipoproteins (HDL, LDL and VLDL) revealed that both LCMUFA isomer diets resulted in similar potentially beneficial alterations in proteins involved in complement activation, blood coagulation, and lipid metabolism. Several lipoprotein proteome changes were significantly correlated with atherosclerotic plaque reduction. CONCLUSIONS: Dietary supplementation with the LCMUFA isomers C20:1 or C22:1 was equally effective in reducing atherosclerosis in LDLr-/-mice and this may partly occur through activation of the Ppar signaling pathways and favorable alterations in the proteome of lipoproteins.

The Yin-Yang Dynamics of DNA Methylation Is the Key Regulator for Smooth Muscle Cell Phenotype Switch and Vascular Remodeling.

OBJECTIVE: DNA methylation plays an important role in chronic diseases such as atherosclerosis, yet the mechanisms are poorly understood. The objective of our study is to indicate the regulatory mechanisms of DNA methylation in vascular smooth muscle cells (VSMCs) and its roles in atherosclerosis. APPROACH AND RESULTS: In ApoE-/- mice fed a Western diet, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, significantly attenuated atherosclerotic lesions (20.1±2.2% versus 30.8±7.5%; P=0.016) and suppressed DNA methyltransferase activity and concomitantly decreased global 5-methylcytosine content in atherosclerotic lesions of ApoE-/- mice. Using a carotid ligation model, we found that 5-aza-2'-deoxycytidine also dramatically inhibited neointimal formation (intimal area: 2.25±0.14×104 versus 4.07±0.22×104 µm2; P<0.01). Abnormal methylation status at the promoter of ten-eleven translocation 2, one of the key demethylation enzymes in mammals, was ameliorated after 5-aza-2'-deoxycytidine treatment, which in turn caused an increase in global DNA hydroxymethylation and 5-hydroxymethylcytosine enrichment at the promoter of Myocardin. In vitro, 5-aza-2'-deoxycytidine treatment or DNA methyltransferase 1 knockdown decreased global 5-methylcytosine content and restored Myocardin expression in VSMCs induced by platelet-derived growth factor, thus preventing excessive VSMCs dedifferentiation, proliferation, and migration. Furthermore, DNA methyltransferase 1 binds to ten-eleven translocation 2 promoter and is required for ten-eleven translocation 2 methylation in VSMCs. CONCLUSIONS: The inhibitory effects of DNA demethylation on global 5-methylcytosine content and ten-eleven translocation 2 hypermethylation in atherosclerotic aorta can recover 5-hydroxymethylcytosine enrichment at the Myocardin promoter and prevent VSMC dedifferentiation and vascular remodeling.

Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced ApoE-/- mice by improving endothelial dysfunction.

BACKGROUND AND AIMS: Chinese dragon's blood has been used to treat blood stasis for thousands of years. Its total phenolic extract (Longxuetongluo capsule, LTC) is used for the treatment of ischemic stroke; however, its protective effect against atherosclerosis remains poorly understood. This paper aims to investigate the antiatherosclerotic effect of LTC and the underlying mechanisms in high-fat diet (HFD)-induced ApoE-/- mice. METHODS: The levels of plasma lipid and areas of atherosclerotic lesions in the aortic sinus in ApoE-/- mice were evaluated. The effect of LTC on the nitric oxide (NO) production in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs) was determined. The adhesion of monocytes to ox-LDL-stimulated HUVECs was further studied. RESULTS: LTC at low, medium, and high doses markedly decreased the atherosclerotic lesion areas of the aortic sinus in HFD-induced ApoE-/- mice by 26.4% (p < 0.05), 30.1% (p < 0.05), and 46.5% (p < 0.01), respectively, although it did not improve the dyslipidemia. Furthermore, LTC restored the diminished NO production of ox-LDL-stimulated HUVECs (p < 0.001) and inhibited the adhesion between monocytes and endothelial cells (p < 0.01). LTC appeared to alleviate ox-LDL-stimulated dysfunction of HUVECs, and inhibit the adhesion of monocytes to HUVECs via the MAPK/IKK/IκB/NF-κB signaling pathway, thus decrease atherosclerotic lesions in the aortic sinus in HFD-induced ApoE-/- mice. CONCLUSIONS: These findings suggest the potential of LTC for use as an effective agent against atherosclerosis.

The intervention effect of zinc supplementation on irbesartan treatment for atherosclerosis of ApoE-/- mice.

To explore the influence of zinc supplementation on irbesartan treatment for atherosclerosis of ApoE gene-deleted mice. Atherosclerosis model mice induced by normal feed were randomly divided into ApoE-/- control group, irbesartan group and zinc sulfate+ irbesartan group, 6 mice each group; C57BL/6J mice with normal feed were regarded as blank control group (n=6). Blank control group and ApoE-/- control group were not given any medical treatment, and irbesartan group were treated with irbesartan (50mg/kg/d), and zinc sulfate+irbesartan group were given zinc sulfate (25mmol/L) treatment besides the administration of irbesartan group. The blood pressure of each mouse was recorded and the blood lipid level was detected after 15 weeks' treatment. The internal inflammatory reaction of the mice was evaluated according to IL-6 and TNF-α level. The oxidative stress was evaluated according to MDA and SOD levels. And the atherosclerosis plaque was analyzed with immunohistochemistry. After 15 weeks of drug administration, it showed that the total cholesterol level, LDL-C, HDL-C and blood pressure level (P<0.05) of the mice were improved significantly with the administration of zinc sulfate+irbesartan compared with irbesartan group, and the oxidative stress response and inflammatory reaction of mice in zinc sulfate+irbesartan group decreased more significantly than those of irbesartan group (P<0.05). In the detection of atherosclerosis plaque, the ratio of plaque area and tube area as well as the improvement degree of mean aortic IMT in zinc sulfate+irbesartan group were superior to those of irbesartan group (P<0.05). Zinc supplementation has certain therapeutic effect on the advanced atherosclerosis of ApoE gene-deleted mice, which can significantly improve the efficacy of irbesartan.