Gene expression in atherosclerotic lesion of ApoE deficient mice

BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.     

Differential gene expression of livers from ApoE deficient mice

A genomic survey for differentially expressed genes was performed to livers of ApoE deficient mice using human cDNA microarray containing approximately 9,000 human cDNA clones. Due to the homology between mouse and human, hybridization was performed at lower stringency condition, 10 degrees below the regular hybridizing temperature. Gene expression profiles of livers corresponding to high levels of blood cholesterol were generated at genomic scale. Thirty-seven genes were randomly selected from a pool of differentially expressed genes and subjected to semi-quantitative RT-PCR, further confirmed the result from microarray hybridization. These included genes associated with atherosclerosis, and novel genes that implied novel pathways correlated to high levels of blood cholesterol. It is promising using human cDNA microarray, the most complete collection among all species, to study other mammalian systems with satisfying speed and accuracy.     

SARI prevents ocular angiogenesis and inflammation in mice

SARI (Suppressor of AP-1, regulated by IFN-β) is known to play an important role in some systemic disease processes such an inflammatory conditions and cancer. We hypothesize that SARI may also play a role in ocular diseases involving inflammation and neovascularization. To explore our hypothesis, further, we investigated an endotoxin-induced uveitis (EIU) and experimental argon laser-induced choroidal neovascularization (CNV) model in SARI wild-type (SARI^WT) and SARI-deficient (SARI^−/−) mice. Through imaging, morphological and immunohistochemical (IHC) studies, we found that SARI deficiency exacerbated the growth of CNV. More VEGF-positive cells were presented in the retina of SARI^−/− mice with CNV. Compared to SARI^WT mice, more inflammatory cells infiltrated the ocular anterior segment and posterior segments in SARI^−/− mice with EIU. Collectively, the results point to a potential dual functional role of SARI in inflammatory ocular diseases, suggesting that SARI could be a potential therapy target for ocular inflammation and neovascularization.     

Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme

Mast cell activation, or neurogenic inflammation, is known to induce lowering of interstitial fluid pressure (P(if)) and plasma protein extravasation (PPE) in several tissues from both rats and mice. To examine a possible role of connective tissue mast cells (CTMCs) in these inflammatory responses, we used mice with dysfunctional CTMCs due to lack of the N-deacetylase/N-sulfotransferase-2 enzyme (NDST-2(-/-)). P(if) and PPE were measured after challenge with compound 48/80 (C48/80), and P(if) alone was measured after treatment either with capsaicin, substance P (SP), or calcitonin gene-related peptide (CGRP). Measurements of P(if) in anesthetized (fentanyl/fluanison and midazolam, 1:1) mice were performed in paw skin with glass capillaries connected to a servo-controlled counterpressure system. PPE was measured with microdialysis by using hollow plasmapheresis fibers (cutoff at 3,000 kDa) placed subcutaneously on the back. Intravenous administration of C48/80 lowered P(if) significantly (P < 0.05) in NDST-2(-/-) mice (-1.67 +/- 0.42 mmHg) compared with vehicle (-0.57 +/- 0.17 mmHg) but the lowering was significantly (P < 0.05) less compared with that of the NDST-2(+/+) mice (-2.31 +/- 0.47 mmHg). PPE was increased 300% after treatment with C48/80 in NDST-2(+/+) mice, whereas there was no increase in PPE in NDST-2(-/-) mice. Capsaicin, SP, and CGRP lowered P(if) significantly (P < 0.05) compared with vehicle and to the same extent in both NDST-2(+/+) and NDST-2(-/-) mice. We can conclude that although NDST-2(-/-) mice demonstrate an altered response in P(if) after mast cell activation, there was no similar alteration after neurogenic inflammation. Therefore, we suggest that neurogenic inflammation in mouse skin is not exclusively dependent on intact CTMCs.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 μg/kg) or paricalcitol (0.1 μg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 μm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-β1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-β expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).     

Melatonin prevents allergic airway inflammation in epicutaneously sensitized mice

Purpose: The pathological process of atopic dermatitis (AD) progressing into other types of allergic diseases such as asthma and allergic rhinitis during the first several years of life is often referred to as the atopic march. Although the phenomenon of atopic march has been recognized for decades, how asthma stems from AD is still not fully understood, confounding a universal strategy to effectively protect people from the atopic march. Methods: We established experimental atopic march mice by first inducing allergic dermatitis with 0.5% fluorescein isothiocyante (FITC) applied to the skin, followed by an ovalbumin (OVA) airway challenge. In addition, by examining serum immunoglobulin (Ig) concentrations, airway cytokines, the levels of oxidative stress markers, histopathological changes in lung tissue and airway hyperresponsiveness (AHR), we were able to validate the successful establishment of the model. Furthermore, by detecting the attenuating effects of melatonin (MT) and the levels of oxidative stress in the atopic march mice, we explored the potential molecular mechanisms involved in the development of atopic march. Results: By successfully establishing an experimental atopic march mouse model, we were able to demonstrate that overproduction of oxidative stress in the lung significantly up-regulated the activation of nuclear factor-κB (NF-κB) signaling pathways causing thymic stromal lymphopoietin (TSLP) release, which further promotes the development of atopic march. Conclusions: To mitigate the development of the atopic march, antioxidants such as MT may be imperative to inhibit NF-κB activation in the lung, especially after the onset of AD.     

Rescuing infusion of miRNA-1 prevents cardiac remodeling in a heart-selective miRNA deficient mouse

Objective

The decreased expression of muscle-specific microRNA-1 (miR-1) has been found in many cardiovascular diseases and is considered to contribute to heart failure (HF). Here we investigated the role of miR-1 in myocardium protection by infusion of miR-1 in a cardiac global miRNA-deficient mouse.

Modulation of allergic inflammation in mice deficient in TNF receptors

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.     

Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2^−/− mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2^−/− mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2^−/− pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2^−/− mice and further defines the sequential steps in lymphatic vessel remodeling.     

Hepatic lysosomal acid lipase drives the autophagy-lysosomal response and alleviates cholesterol metabolic disorder in ApoE deficient mice

Lysosomal acid lipase (LAL)-dependent lipolysis degrades cholesteryl ester (CE) and triglyceride in the lysosome. LAL deficiency in human and mice leads to hypercholesterolemia, hepatic CE deposition, and atherosclerosis. Despite its hepatocyte-specific deficiency leads to CE accumulation, the regulation of LAL in cholesterol metabolic disease remains elusive. For the in vitro study, the target gene Lipa was transfected with recombinant shRNA or lentiviral vector in Hepa1-6 cells. It was found that LAL silencing in cells affected lysosomal function by reducing LAL activity and proteolytic activity, and altered the expression of genes related to cholesterol metabolism and autophagy, leading to cholesterol accumulation; whereas LAL overexpression improved the above effects. To explore the impacts of hepatic LAL on cholesterol metabolic disease in vivo, apolipoprotein E deficient (ApoE^−/−) mice were intravenously injected with lentivirus to achieve hepatic LAL overexpression and fed a Western diet for 16 weeks. The results showed that hepatic LAL overexpression significantly reduced plasma lipid levels, alleviated inflammation and oxidative status in plasma and liver, and attenuated hepatic steatosis and fibrosis in ApoE^−/− mice. Mechanically, hepatic LAL promoted cholesterol transport and biliary excretion by increasing liver X receptor alpha (LXRα) and its downstream genes, and modulated the compliance of the autophagy-lysosomal pathway. Our data provide the original evidence of the validity of hepatic LAL in controlling cholesterol metabolism and liver homeostasis, suggesting that targeting hepatic LAL may provide a promising approach to rescue cholesterol metabolic disorders, such as hypercholesterolemia and liver disease.     

Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

We investigated the effect of tissue kallikrein infusion on cardiac protection at acute and sub-acute phases after myocardial infarction (MI). Immediately after MI, rats were infused with purified tissue kallikrein, with or without icatibant (a kinin B2 receptor antagonist). Intramyocardial injection of kallikrein reduced myocardial infarct size and inhibited cardiomyocyte apoptosis at 1 day after MI associated with increased nitric oxide levels, Akt and glycogen synthase kinase-3beta phosphorylation and decreased caspase-3 activation. Kallikrein infusion for 7 days improved cardiac function, normalized left ventricular wall thickness and decreased monocyte/macrophage infiltration in the infarct heart. Kallikrein treatment reduced NADH oxidase expression and activity, superoxide formation and malondialdehyde levels, and reduced MAPK and Ikappa-Balpha phosphorylation, NF-kappaB activation and MCP-1 and VCAM-1 expression. Kallikrein's effects were all blocked by icatibant. These results indicate that kallikrein through kinin B2 receptor activation prevents apoptosis, inflammation and ventricular remodeling by increased nitric oxide formation and suppression of oxidative stress-mediated signaling pathways.     

Endurance training prevents inflammation and apoptosis in hypothalamic neurons of obese mice

This study investigated the effects of exercise training in regulating inflammatory processes, endoplasmic reticulum stress, and apoptosis in hypothalamic neurons of obese mice. Swiss mice were distributed into three groups: Lean mice (Lean), sedentary animals fed a standard diet; obese mice (Obese), sedentary animals fed a high-fat diet (HFD); trained obese mice (T. Obese), animals fed with HFD and concurrently subjected to an endurance training protocol for 8 weeks. In the endurance training protocol, mice ran on a treadmill at 60% of peak workload for 1 hr, 5 days/week for 8 weeks. Twenty-four hours after the last exercise session, the euthanasia was performed. Western blot, quantitative real-time polymerase chain reaction, and terminal deoxynucleotide transferase biotin-dUTP nick end-labeling (TUNEL) techniques were used for the analysis of interest. The results show exercise training increased phosphorylation of leptin signaling pathway proteins (pJAK2/pSTAT3) and reduced the content of tumor necrosis factor α, toll-like receptor 4, suppressor of cytokine signaling 3, protein–tyrosine phosphatase 1B as well as the phosphorylation of IkB kinase in the hypothalamus of T. Obese animals. A reduction of macrophage activation and phosphorylation of eukaryotic initiation factor 2α, and protein kinase RNA-like endoplasmic reticulum kinase (PERK) were also observed in exercised animals. Furthermore, exercise decreased the expression of the proapoptotic protein (PARP1) and increased anti-inflammatory (IL-10) and antiapoptotic (Bcl2) proteins. Using the TUNEL technique, we observed that the exercised animals had lower DNA fragmentation. Finally, physical exercise preserved pro-opiomelanocortin messenger RNA content. In conclusion, exercise training was able to reorganize the control of the energy balance through anti-inflammatory and antiapoptotic responses in hypothalamic tissue of obese mice.     

Urokinase plasminogen activator receptor promotes macrophage infiltration into the vascular wall of ApoE deficient mice

The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.     

IL-7R blockade reduces post-myocardial infarction-induced atherosclerotic plaque inflammation in ApoE−/- mice

Modulating inflammation by targeting IL-1β reduces recurrent athero-thrombotic cardiovascular events without lipid lowering. This presents an opportunity to explore other pathways associated with the IL-1β signaling cascade to modulate the inflammatory response post-myocardial infarction (MI). IL-7 is a mediator of the inflammatory pathway involved in monocyte trafficking into atherosclerotic plaques and levels of IL-7 have been shown to be elevated in patients with acute MI. Recurrent athero-thrombotic events are believed to be mediated in part by index MI-induced exacerbation of inflammation in atherosclerotic plaques. The objective of the study was to assess the feasibility of IL-7R blockade to modulate atherosclerotic plaque inflammation following acute MI in ApoE^?/- mice. Mice were fed Western diet for 12 weeks and then subjected to coronary occlusion to induce an acute MI. IL-7 expression was determined using qRT-PCR and immuno-staining, and IL-7R was assessed using flow cytometry. Plaque inflammation was evaluated using immunohistochemistry. IL-7R blockade was accomplished with monoclonal antibody to IL-7R. IL-7 mRNA expression was significantly increased in the cardiac tissue of mice subjected to MI but not in controls. IL-7 staining was observed in the coronary artery. Plaque macrophage and lipid content were significantly increased after MI. IL-7R antibody treatment but not control IgG significantly reduced macrophage and lipid content in atherosclerotic plaques. The results show that IL-7R antibody treatment reduces monocyte/macrophage and lipid content in the atherosclerotic plaque following MI suggesting a potential new target to mitigate increased plaque inflammation post-MI.     

Gas6 deficiency prevents liver inflammation, steatohepatitis, and fibrosis in mice

The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in β-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1β, TNF-α, lymphotoxin-β, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-β and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.     

Overexpression of apolipoprotein AV in the liver reduces plasma triglyceride and cholesterol but not HDL in ApoE deficient mice

It has been shown that adenovirus-mediated overexpression of human ApoAV (hApoAV) in C57BL/6 mice results in decreased plasma triglyceride (TG) and total cholesterol (TC) levels with a major reduction occurring in the HDL fraction. In order to study the effect of ApoAV on hypercholesterolemic mice, an adenoviral vector expressing hApoAV was constructed and injected into ApoE deficient mice. High levels of hApoAV mRNA in the liver and ApoAV proteins in the liver and plasma were detected. The treatment reduced plasma TG levels by 50% and 75%, and TC levels by 45% and 58% at day 3 and 7, respectively, after treatment as compared with a control group treated with Ad-hAP (human alkaline phosphatase). Plasma HDL-C levels remained unaltered, which were different from normolipidemic mice. These findings suggest that ApoAV might serve as a therapeutic agent for hyperlipidemic disorder.     

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.