Development Of Atherosclerosis In ApoE Research Articles

Abstract 568: ApoE-/- Mice Lacking Hemopexin Develop Aggravated Atherosclerosis via Mechanisms That Include Altered High Density Lipoprotein Inflammatory Properties and Macrophage Function

Introduction: The etiology of atherosclerosis is multi-faceted. Atherosclerotic plaques are characterized by the accumulation of lipid-laden macrophages, reactive oxygen species (ROS) and pro-inflammatory lipoproteins. Recent studies suggest that the anti- or pro-inflammatory nature of high density lipoprotein (HDL) may be a more sensitive indicator of atherosclerosis than HDL-cholesterol (HDL-C) levels alone. Our laboratory previously reported that hemopexin (Hx) is significantly increased and associated with pro-inflammatory HDL under atherogenic conditions. Hx is a heme scavenging protein which together with macrophages, plays a role in mitigating heme and ROS mediated oxidative damage. However, the role of Hx in atherosclerosis is not known. Hypothesis: We hypothesized that absence of Hx plays an important role in the accumulation of pro-inflammatory HDL, macrophage function, and the development of atherosclerosis. Methods: We crossed Hx -/- mice onto the apoE -/- background (HxE -/- mice). Chow diet fed HxE -/- and apoE -/- mice (n=10-15) were analyzed for lipoprotein inflammatory properties, macrophage gene expression, and atherosclerosis. Results: Serum ROS and heme (p<0.05) were significantly higher in the HxE -/- mice. Livers of HxE -/- mice showed increased lipid peroxides (p<0.001), and ex vivo monocyte chemotactic assays indicated that HDL from HxE -/- mice was more pro-inflammatory (p<0.002). Expression of macrophage cholesterol efflux genes: ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) and its transcriptional regulator liver X receptor-alpha (LXRα), were significantly reduced (p<0.05) whereas expression of the classical M1 macrophage cell-surface receptor CCR2 was significantly increased (p=0.005) in the HxE -/- mice. Atherosclerotic plaque area and macrophage infiltration was significantly higher in the aortic sinus (p<0.004), and atherosclerotic lesions in the aortas were significantly higher in the HxE -/- mice (p<0.05). Conclusion: Our results suggest for the first time that Hx plays a protective role in the inflammatory properties of HDL, macrophage function and in the development of atherosclerosis in apoE-/- mice. We conclude that Hx is a novel target for the treatment of atherosclerosis.

IL-9: a new culprit in atherosclerosis?

This editorial refers to ‘IL-9 aggravates the development of atherosclerosis in ApoE−/− mice’ by W. Zhang et al. , pp. 453–464. Atherosclerosis is a chronic inflammatory disease of the arterial wall driven by innate and adaptive immunity. Atherosclerotic lesions contain a variety of cells including smooth muscle cells, macrophages, and T lymphocytes, as well as other inflammatory cells, such as mast cells and NKT cells. This collection of cells promotes the development of atherosclerosis through the production of inflammatory factors. Among CD4+T cells, Th 1 cells have been shown to exert proatherogenic effects, whereas regulatory T cells (Treg) display atheroprotective properties and the role of Th2 and Th17 cells remains unclear.1 The differentiation of Th cells into effector subsets that secrete specific pro- or anti-inflammatory cytokines is critical for the initiation and progression of atherosclerosis. The development of Th subpopulations is dependent on the expression of lineage-specific transcription factors that are regulated by specific cell–cell interactions, as well as by the cytokine environment. Among these is the recently described Th9 cell subset that preferentially secretes IL-9.2 IL-9 is a pleiotropic cytokine that is highly expressed in diseases where Th2 cytokines, including IL-4, IL-5, and IL-13, are up-regulated, which explains why it was initially considered as a Th2 cytokine. In addition to Th9 or Th2 cells, mast cells, eosinophils, innate lymphoid cells (ILCs), and NKT cells have been shown to be sources for this cytokine.3 The differentiation of Th9 cells is dependent on transcription factors that include PU.1, downstream of …

Activation of an Innate Immune Receptor, Nod1, Accelerates Atherogenesis in Apoe−/− Mice

Atherosclerosis is essentially a vascular inflammatory process in the presence of an excess amount of lipid. We have recently reported that oral administration of a nucleotide-binding oligomerization domain (Nod)-1 ligand, FK565, induced vascular inflammation in vivo. No studies, however, have proven the association between Nod1 and atherosclerosis in vivo. To investigate a potential role of NOD1 in atherogenesis, we orally administered FK565 to apolipoprotein E knockout (Apoe(-/-)) mice for 4 wk intermittently and performed quantification of atherosclerotic lesions in aortic roots and aortas, immunohistochemical analyses, and microarray-based gene expression profiling of aortic roots. FK565 administration accelerated the development of atherosclerosis in Apoe(-/-) mice, and the effect was dependent on Nod1 in non-bone marrow origin cells by bone marrow transplantation experiments. Immunohistochemical studies revealed the increases in the accumulation of macrophages and CD3 T cells within the plaques in aortic roots. Gene expression analyses of aortic roots demonstrated a marked upregulation of the Ccl5 gene during early stage of atherogenesis, and the treatment with Ccl5 antagonist significantly inhibited the acceleration of atherosclerosis in FK565-administered Apoe(-/-) mice. Additionally, as compared with Apoe(-/-) mice, Apoe and Nod1 double-knockout mice showed reduced development of atherosclerotic lesions from the early stage as well as their delayed progression and a significant reduction in Ccl5 mRNA levels at 9 wk of age. Data in the present study show that the Nod1 signaling pathway in non-bone marrow-derived cells contributes to the development of atherosclerosis.

Transplantation of periaortic adipose tissue from angiotensin receptor blocker-treated mice markedly ameliorates atherosclerosis development in apoE-/- mice.

Perivascular adipose tissue is implicated in vasoreactivity; however, its effect on atherosclerosis remains undefined. We examined the effect of a high-cholesterol diet (HCD) on phenotypic alterations of the thoracic periaortic adipose tissue (tPAT) in apoE-deficient (apoE(-/-)) mice. Gene expression of the components of the renin angiotensin system and that of macrophage markers were significantly higher in apoE(-/-) mice fed an HCD than in those fed a chow diet (CD). These changes were absent both in angiotensin II (AngII) receptor blocker (ARB)-treated apoE(-/-) mice and in Ang II type 1 (AT1) receptor-deficient apoE(-/-) (Agtr1(-/-)/apoE(-/-)) mice. To evaluate their effect on atherosclerosis, we transplanted tPAT into apoE(-/-) mice alongside the distal abdominal aorta. Transplanted tPAT was harvested from apoE(-/-) and Agtr1(-/-)/apoE(-/-) mice fed a CD (tPAT-CD/apoE(-/-), tPAT-CD/Agtr1(-/-)/apoE(-/-)), HCD (tPAT-HCD/apoE(-/-), tPAT-HCD/Agtr1(-/-)/apoE(-/-)), or HCD in combination with ARB treatment (tPAT-HCD/ARB/apoE(-/-)). Four weeks after transplantation, a significantly increased oil red O-positive area was observed in the aorta of tPAT-HCD/apoE(-/-) mice than in tPAT-CD/apoE(-/-) mice. Such a change was absent in tPAT-HCD/ARB/apoE(-/-) and tPAT-HCD/Agtr1(-/-)/apoE(-/-) mice. Our findings demonstrated that AT1 receptor plays a crucial role in HCD-induced phenotypic alterations of tPAT, modulation of which could exert beneficial effects on atherosclerosis.

Chlorogenic acid protects against atherosclerosis in ApoE-/- mice and promotes cholesterol efflux from RAW264.7 macrophages.

Chlorogenic acid (CGA) is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE−/− mice and its potential mechanism. ApoE−/− mice were fed a cholesterol-rich diet without (control) or with CGA (200 and 400 mg/kg) or atorvastatin (4 mg/kg) for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg) reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg) also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL)-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE−/− mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA.

Abstract 68: Activation of Brown Adipose Tissue Reduces Development of Atherosclerosis

Objectives: Brown adipose tissue (BAT) recently emerged as a novel player in lipoprotein metabolism. BAT combusts high amounts fatty acids into heat resulting in reduced plasma triglyceride (TG) levels as well as obesity. However, the precise role of BAT in cholesterol metabolism and atherosclerosis development remains unclear. We aimed to assess the effect of stimulating BAT activity by ß3-adrenergic receptor (ß3-AR) agonism on lipoprotein metabolism and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism. Methods and results: Mice were fed a Western-type diet to induce dyslipidemia and were treated subcutaneously with the selective ß3-AR agonist CL316243 (3x20 μg/week) or vehicle for 10 weeks. ß3-AR agonism reduced total fat mass (-59%) and white adipose tissue (WAT) pad size (-40%). ß3-AR agonism decreased the size of intracellular lipid vacuoles in BAT, and increased UCP-1 expression in WAT, indicating activation of brown adipocytes ( i.e. BAT) as well as their precursors ( i.e. WAT). These effects were accompanied by increased uptake of glycerol tri[ 3 H]oleate from VLDL-like particles by BAT (+125%) and by WAT (+120%) as well as fatty acid oxidation (+25%) as determined by indirect calorimetry. In line with these data, activation of BAT decreased plasma levels of TG (-46%) and (V)LDL-cholesterol (-24%) and increased HDL-cholesterol (+23%), accompanied by increased reverse cholesterol transport (+2.5-fold). As a consequence of improving lipoprotein metabolism, BAT activation reduced atherosclerosis development (-43%) as well as lesion severity, indicated by more mild type I-III lesions (+64%) and reduced severe type IV-V lesions (-66%), in the aortic root. These effects were dependent on a functional hepatic apoE-LDLr clearance pathway, as BAT activation in apoe -/- and ldlr -/- mice lowered plasma TG levels but did not attenuate hypercholesterolemia and atherosclerosis Conclusion: We demonstrate that activation of BAT via the ß3-AR is a powerful tool to reduce dyslipidemia and protect against atherosclerosis. These findings indicate that activation of BAT is a promising novel strategy to combat dyslipidemia and cardiovascular disease.

Is hyperuricemia a risk factor for arteriosclerosis? Uric acid and arteriosclerosis in apolipoprotein e-deficient mice.

Although hyperlipidemia, high blood pressure, and diabetes increase the risk of arteriosclerosis, it is not clear whether hyperuricemia increases the risk of arteriosclerosis or not. We examined the effects of uric acid and curative drugs for hyperuricemia on atherosclerosis-susceptible C57BL/6J apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (age: 6 weeks) were fed a normal diet (normal diet group) or a uric acid-enriched diet. Mice fed the uric acid-enriched diet were divided into three groups and administered a drinking vehicle (high uric acid diet group), allopurinol (20 mg·kg(-1)·d(-1)), or benzbromarone (20 mg·kg(-1)·d(-1)) for 10 weeks. Serum uric acid concentrations were higher in the high uric acid diet group than in the normal diet group, and concentrations in the allopurinol and benzbromarone groups were lower than in the high uric acid diet group. Serum total cholesterol and triglyceride levels were lower in the allopurinol group than in the high uric acid diet group. Oxidative stress was lower in the benzbromarone group than in the high uric acid diet group. Atherosclerotic lesion areas were smaller in the allopurinol and benzbromarone groups than in the high uric acid diet group. Thus, hyperuricemia may not be an independent risk factor for arteriosclerosis; however, the administration of allopurinol and benzbromarone prevented the development of atherosclerosis in apoE(-/-) mice fed a uric acid-enriched diet. The anti-atherosclerotic effect was in part due to lower total cholesterol and oxidative stress in the serum. Other possible mechanisms underlying this effect should be investigated.

Metoprolol reduces proinflammatory cytokines and atherosclerosis in ApoE-/- mice.

A few studies in animals and humans suggest that metoprolol (β1-selective adrenoceptor antagonist) may have a direct antiatherosclerotic effect. However, the mechanism behind this protective effect has not been established. The aim of the present study was to evaluate the effect of metoprolol on development of atherosclerosis in ApoE−/− mice and investigate its effect on the release of proinflammatory cytokines. Male ApoE−/− mice were treated with metoprolol (2.5 mg/kg/h) or saline for 11 weeks via osmotic minipumps. Atherosclerosis was assessed in thoracic aorta and aortic root. Total cholesterol levels and Th1/Th2 cytokines were analyzed in serum and macrophage content in lesions by immunohistochemistry. Metoprolol significantly reduced atherosclerotic plaque area in thoracic aorta (P < 0.05 versus Control). Further, metoprolol reduced serum TNFα and the chemokine CXCL1 (P < 0.01 versus Control for both) as well as decreasing the macrophage content in the plaques (P < 0.01 versus Control). Total cholesterol levels were not affected. In this study we found that a moderate dose of metoprolol significantly reduced atherosclerotic plaque area in thoracic aorta of ApoE−/− mice. Metoprolol also decreased serum levels of proinflammatory cytokines TNFα and CXCL1 and macrophage content in the plaques, showing that metoprolol has an anti-inflammatory effect.

The Effect of PPE-Induced Emphysema and Chronic LPS-Induced Pulmonary Inflammation on Atherosclerosis Development in APOE*3-LEIDEN Mice

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, airways obstruction and emphysema, and is a risk factor for cardiovascular disease (CVD). However, the contribution of these individual COPD components to this increased risk is unknown. Therefore, the aim of this study was to determine the contribution of emphysema in the presence or absence of pulmonary inflammation to the increased risk of CVD, using a mouse model for atherosclerosis. Because smoke is a known risk factor for both COPD and CVD, emphysema was induced by intratracheal instillation of porcine pancreatic elastase (PPE).MethodsHyperlipidemic APOE*3-Leiden mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and after 4 weeks, mice received a Western-type diet (WTD). To study the effect of emphysema combined with pulmonary inflammation on atherosclerosis, mice received 30 µg PPE and during WTD feeding, mice were intranasally instilled with vehicle or low-dose lipopolysaccharide (LPS; 1 µg/mouse, twice weekly). After 20 weeks WTD, mice were sacrificed and emphysema, pulmonary inflammation and atherosclerosis were analysed.ResultsIntratracheal PPE administration resulted in a dose-dependent increase in emphysema, whereas atherosclerotic lesion area was not affected by PPE treatment. Additional low-dose intranasal LPS administration induced a low-grade systemic IL-6 response, as compared to vehicle. Combining intratracheal PPE with intranasal LPS instillation significantly increased the number of pulmonary macrophages and neutrophils. Plasma lipids during the study were not different. LPS instillation caused a limited, but significant increase in the atherosclerotic lesion area. This increase was not further enhanced by PPE.ConclusionThis study shows for the first time that PPE-induced emphysema both in the presence and absence of pulmonary inflammation does not affect atherosclerotic lesion development.

Thrombin generation and atherosclerosis

Atherosclerosis is a major cause of mortality worldwide. The important role inflammation plays in atherosclerosis is evident through the participation of inflammatory cells in the development and progression of the disease. Thrombin is the central protease of the coagulation cascade, involved in the formation of a hemostatic plug to avoid severe bleeding. In addition, thrombin is a key factor in regulating inflammatory processes, signaling through protease activated receptors. We propose that thrombin may be a relevant factor in the atherosclerosis coagulation-inflammation axis. Human histological data show abundant coagulation activity within atherosclerotic lesions with thrombin activity being related to atherosclerotic plaque development and (in)stability. Animal studies establish that the generated thrombin level relates to progression of atherosclerosis, with hypercoagulability producing advanced atherosclerosis in mice with an Apolipoprotein E-deficient (ApoE(-/-)) background. Several studies show that administration of direct oral anticoagulants, like dabigatran and rivaroxaban, attenuate atherosclerosis development in ApoE(-/-) mice. In this review we explore several mechanisms by which thrombin may operate in modifying the chronic process of atherosclerosis. One of the key elements may be the conversion of thrombin, from a physiological regulator of hemostasis towards an inflammation-mediator under pathophysiological conditions, contributing to a switch in the thrombin-activated protein C (APC) regulation. The ongoing inflammatory activity, indicated by the activation of pro-inflammatory cytokines, neutrophils and neutrophil extracellular traps, drive thrombin generation, while diminishing APC formation. The net result is accelerated pro-inflammatory and pro-thrombotic changes in blood and in the vessel wall. We conclude that these atherogenic influences of thrombin may be clinically relevant in the long-term. Further the treatment with long-term anticoagulant therapy deserves further attention as to its potential, vascular side effects.

High-fat diet alters protein composition of detergent-resistant membrane microdomains

A high-lipid diet is one of the main risk factors in atherosclerosis and can induce changes in the composition of plasma membrane microdomains. In response, important functions such as vesicle trafficking, protein docking, signaling and receptor recognition are significantly altered. In particular, interactions of heat-shock proteins (Hsps), acting as danger signals, with components of the membrane microdomains can influence signaling pathways and the inflammatory response of cells. Our study focuses on the composition of detergent-resistant membrane (DRM) isolated from ApoE-/- mice fed a standard or high-fat diet with and without fluvastatin treatment versus appropriate controls. Biochemical studies, immunoblotting and liquid chromatography mass spectrometric analysis were performed to investigate whether the structural components (such as caveolin and cavin) of the detergent-resistant microdomains were correlated with the expression and secretion of stress-inducible Hsps (Hsp70 and Hsp90) and AKT phosphorylation in experimental atherosclerosis. ApoE-/- mice challenged with a high-fat diet developed extensive atherosclerotic plaques in lesion-prone areas. DRM harvested from hyperlipidemic animals showed a modified biochemical composition with cholesterol, glycerolipids, caveolin-1 and phospho-AKT being up-regulated, whereas cavin-1 and dynamin were down-regulated. The data also demonstrated the co-fractionation of Hsps with caveolin-1 in isolated DRM, expression being positively correlated with their secretion into blood serum. Statin therapy significantly attenuated the processes induced by the development of atherosclerosis in ApoE-/- mice under a high-fat diet. Thus, high-lipid stress induces profound changes in DRM biochemistry and modifies the cellular response, supporting the systemic inflammatory onset of atherosclerosis.

Inhibition of Endoplasmic Reticulum Stress and Atherosclerosis by 2-Aminopurine in Apolipoprotein E-Deficient Mice

We previously reported that the apolipoprotein (apo) B48-carrying lipoproteins obtained from apoE knockout (apoE −/−) mice, so called E−/B48 lipoproteins, transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2α (eIF-2α). Furthermore, the eIF-2α phosphorylation inhibitor, 2-aminopurine (2-AP), attenuated E−/B48 lipoprotein-induced foam cell formation. The present report studied the effect of 2-AP on atherosclerosis in apoE −/− mice. Our results showed that the level of food intake, bodyweight, plasma cholesterol, and triglycerides was comparable in apoE −/− mice treated with or without 2-AP. However, the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-AP-treated apoE −/− mice were reduced by about 55% and 39%, respectively, compared to samples from untreated control apoE −/− mice. In addition, the 2-AP-treated apoE −/− mice showed a significant decrease in glucose-regulated protein 78 (GRP78) and phosphorylated eIF-2α in their aortic samples as compared to levels in untreated control apoE −/− mice. These observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoE −/− mice and that therapeutic strategies can be developed for using eIF-2α phosphorylation inhibitors, such as 2-AP, to prevent or treat atherosclerosis.

Inhibition of monocyte chemotaxis by VB-201, a small molecule lecinoxoid, hinders atherosclerosis development in ApoE−/− mice

ObjectiveMonocytes are motile cells which sense inflammatory stimuli and subsequently migrate to sites of inflammation. Key players in host defense, monocytes have nevertheless been implicated as requisite mediators of several chronic inflammatory diseases. Inhibition of monocyte chemotaxis is therefore an attractive anti-inflammatory strategy. Oxidized phospholipids (OxPL) are native regulators of inflammation, yet their direct effect on monocyte chemotaxis is poorly defined. In this study, we investigated the direct effect of natural and synthetic phospholipids on monocyte chemotaxis. MethodsExploring various phospholipids using in vitro chemotaxis assays, we found that the natural phospholipid 1-palmitoyl-2-glutaryl phosphatidylcholine (PGPC) can decrease monocyte chemotaxis by 50%, while other tested OxPL had no effect. We generated a library of synthetic OxPL designated lecinoxoids, which was screened for anti-inflammatory properties. Results and conclusionsVB-201, a small-molecule lecinoxoid, exhibited up to 90% inhibition of monocyte chemotaxis in vitro. Molecular analysis revealed that the effect of VB-201 was not restricted to a specific chemotactic ligand or receptor, and resulted from inhibition of signaling pathways required for monocyte chemotaxis. Interestingly, VB-201 did not inhibit monocyte adhesion or phagocytosis and had no effect on chemotaxis of CD4+ T-cells or neutrophils. In vivo, oral treatment with VB-201 reduced monocyte migration in a peritonitis model and inhibited atheroma development in ApoE−/− mice, without affecting cholesterol or triglyceride levels. Our findings highlight a novel role played by native and synthetic phospholipids in regulation of monocyte chemotaxis. The data strengthen the involvement of phospholipids as key signaling molecules in inflammatory settings and demonstrate their potential therapeutic applicability.

Niacin Reduces Atherosclerosis Development in APOE*3Leiden.CETP Mice Mainly by Reducing NonHDL-Cholesterol

ObjectiveNiacin potently lowers triglycerides, mildly decreases LDL-cholesterol, and largely increases HDL-cholesterol. Despite evidence for an atheroprotective effect of niacin from previous small clinical studies, the large outcome trials, AIM-HIGH and HPS2-THRIVE did not reveal additional beneficial effects of niacin (alone or in combination with laropiprant) on top of statin treatment. We aimed to address this apparent discrepancy by investigating the effects of niacin without and with simvastatin on atherosclerosis development and determine the underlying mechanisms, in APOE*3Leiden.CETP mice, a model for familial dysbetalipoproteinemia (FD).Approach and ResultsMice were fed a western-type diet containing cholesterol without or with niacin (120 mg/kg/day), simvastatin (36 mg/kg/day) or their combination for 18 weeks. Similarly as in FD patients, niacin reduced total cholesterol by -39% and triglycerides by −50%, (both P<0.001). Simvastatin and the combination reduced total cholesterol (−30%; −55%, P<0.001) where the combination revealed a greater reduction compared to simvastatin (−36%, P<0.001). Niacin decreased total cholesterol and triglycerides primarily by increasing VLDL clearance. Niacin increased HDL-cholesterol (+28%, P<0.01) and mildly increased reverse cholesterol transport. All treatments reduced monocyte adhesion to the endothelium (−46%; −47%, P<0.01; −53%, P<0.001), atherosclerotic lesion area (−78%; −49%, P<0.01; −87%, P<0.001) and severity. Compared to simvastatin, the combination increased plaque stability index [(SMC+collagen)/macrophages] (3-fold, P<0.01). Niacin and the combination reduced T cells in the aortic root (−71%, P<0.01; −81%, P<0.001). Lesion area was strongly predicted by nonHDL-cholesterol (R2 = 0.69, P<0.001) and to a much lesser extent by HDL-cholesterol (R2 = 0.20, P<0.001).ConclusionNiacin decreases atherosclerosis development mainly by reducing nonHDL-cholesterol with modest HDL-cholesterol-raising and additional anti-inflammatory effects. The additive effect of niacin on top of simvastatin is mostly dependent on its nonHDL-cholesterol-lowering capacities. These data suggest that clinical beneficial effects of niacin are largely dependent on its ability to lower LDL-cholesterol on top of concomitant lipid-lowering therapy.

Vascular angiotensin II type 2 receptor attenuates atherosclerosis via a kinin/NO-dependent mechanism

The angiotensin II (Ang II) type 1 receptor exerts pro-atherogenic action by augmenting oxidative stress, whereas the Ang II type 2 receptor (AT2)-mediated effect on atherosclerosis remains controversial. AT2 transgenic (AT2-Tg) mice, which overexpress AT2 in their vascular smooth muscle cells, were crossed with apoE-deficient (apoE(-/-)) mice to generate AT2 transgenic apoE(-/-) mice (AT2-Tg/apoE(-/-)). A subpressor dose of Ang II infusion exaggerated atherosclerosis development in apoE(-/-) mice, which was markedly suppressed in AT2-Tg/apoE(-/-) mice. Inhibitors of nitric oxide (NO) synthase (L-NAME) or bradykinin type 2 receptor completely abolished AT2-mediated anti-atherogenic actions. The vascular cell adhesion molecule-1 expression levels and degree of monocyte/macrophage accumulation in the intima were also considerably reduced in AT2-Tg/apoE(-/-) mice; these phenomena were completely reversed by L-NAME treatment. Ang II infusion significantly enhanced the accumulation of dihydroethidium-positive mononuclear cells in the intima and mRNA expression levels of Nox2, a phagocytic cell-type NADPH oxidase subunit in apoE(-/-) mice, which was completely inhibited in AT2-Tg/apoE(-/-) mice. Vascular AT2 stimulation exerts anti-atherogenic actions in an endothelial kinin/NO-dependent manner, and its anti-oxidative effect is likely to be exerted by inhibiting the accumulation of superoxide-producing mononuclear leukocytes.

Abstract 69: Anacetrapib Dose-dependently Decreases Atherosclerosis Development and Adds to the Beneficial Effects of Atorvastatin in APOE*3Leiden.CETP Mice

Introduction The residual risk of cardiovascular disease that remains after statin treatment has triggered the search for a secondary treatment target. Epidemiological studies propose HDL-cholesterol (HDL-C) as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. In human intervention trials, the CETP inhibitor anacetrapib decreases (V)LDL-C by 30-40% and increases HDL-C by 40-140%. Hypothesis Complete inhibition of CETP activity may result in adverse effects as compared to partial inhibition due to the appearance of a dysfunctional HDL. We, therefore, evaluated the effect of a broad treatment window of anacetrapib-induced CETP inhibition with partial to full inhibition, as well as the combination of atorvastatin and anacetrapib on atherosclerosis development in APOE*3Leiden.CETP mice. Methods Female mice were fed a Western-type diet containing 0.1% cholesterol without or with incremental dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/d), atorvastatin (2.4 mg/kg/d) or a combination of anacetrapib (0.3 mg/kg/d) and atorvastatin (2.4 mg/kg/d) for 20 weeks. Effects on plasma lipids, CETP activity and levels, as well as atherosclerotic lesion size and severity were assessed. Results Anacetrapib dose-dependently reduced CETP activity (-60% to -100%, P&lt;0.001) and increased CETP levels (+13% to +31%, P&lt;0.05), thereby decreasing nonHDL-C (-23% to -44%, P&lt;0.001) and increasing HDL-C (+32% to +88%, P&lt;0.001). Atorvastatin decreased CETP activity (-29%, P&lt;0.001) and nonHDL-C (-36%, P&lt;0.001). Anacetrapib dose-dependently decreased atherosclerotic lesion size (-36%, P&lt;0.05 to -92%, P&lt;0.001) and the percentage severe lesions. Anacetrapib added to the effects of atorvastatin by further reducing nonHDL-C (-36%, P&lt;0.001), increasing HDL-C (+72%, P&lt;0.001) and decreasing lesion size (-86%, P&lt;0.001) and severity. Results on lesion composition are pending. Conclusions Anacetrapib dose-dependently decreases atherosclerosis development and adds to the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. Total blockage of CETP activity does not reveal adverse effects as compared to partial blockage.

Interleukin‐17A plays an important role in endothelial cell activation and atherogenesis in ApoE−/− mice

Interleukin‐17 (IL‐17) cytokines are a family of pro‐inflammatory cytokines. IL‐17A has been implicated in numerous autoimmune and inflammatory diseases including atherosclerosis. However, the role of IL‐17A in early atherogenesis remained to be determined. The objective of our study is to determine the role of IL‐17A in early atherogenesis. Previously, using a bioinformatics approach, we found that IL‐17 receptors A and C, and adaptor protein, ACT1, are expressed in human vascular tissue, suggesting that IL‐17A has a role in chronic vascular inflammation. The data for the expression of these receptors and ACT1 were not available in mouse vascular tissue; however, using immunostaining, we found that IL‐17RA is expressed in mouse aorta. Moreover, plasma levels of IL‐17A are found to be increased in atherogenic ApoE−/− mice compared with wild type mice. IL‐17A treatment was found to induce pro‐inflammatory cytokines production in mouse aortic cells. Furthermore, to elucidate the role of IL‐17A in atherogenesis in ApoE−/− mice, we cross‐bred IL‐17A−/− mice with ApoE−/− mice to generate our double knock‐out mouse model, ApoE−/−IL‐17A−/− mice. Our data suggest that IL‐17A have an important role in early atherogenesis and with our novel mouse model we will be able to elucidate the function as well as the mechanism of IL‐17A in the endothelial cell activation and development of atherosclerosis in ApoE−/− mice.

Resveratrol protects against atherosclerosis, but does not add to the antiatherogenic effect of atorvastatin, in APOE*3-Leiden.CETP mice

Resveratrol is a major constituent of traditional Asian medicinal herbs and red wine and is suggested to be a potential antiatherosclerotic drug due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate whether resveratrol protects against atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice and adds to the antiatherogenic effect of mild statin treatment, currently the most widely used antiatherogenic therapy. E3L.CETP mice were fed a cholesterol-rich diet without (control) or with resveratrol (0.01% w/w), atorvastatin (0.0027% w/w) or both for 14 weeks. During the study plasma lipid, inflammatory and oxidative stress parameters were determined. Resveratrol reduced atherosclerotic lesion area (−52%) in the aortic root, comparable to atorvastatin (−40%) and the combination of both drugs (−47%). The collagen/macrophage ratio in the atherosclerotic lesion, a marker of plaque stability, was increased by resveratrol (+108%), atorvastatin (+124%) and the combination (+154%). Resveratrol decreased plasma cholesterol levels (−19%) comparable to atorvastatin (−19%) and the combination (−22%), which was completely confined to (very)low-density lipoprotein cholesterol levels in all groups. Post hoc analyses showed that the antiatherogenic effect of atorvastatin could be explained by cholesterol lowering, while the antiatherosclerotic effect of resveratrol could be attributed to factors additional to cholesterol lowering. Markers of inflammation and oxidative stress were not different, but resveratrol improved macrophage function. We conclude that resveratrol potently reduces atherosclerosis development and induces a more stable lesion phenotype in E3L.CETP mice. However, under the experimental conditions tested, resveratrol does not add to the antiatherogenic effect of atorvastatin.

Aortic Smooth Muscle Cells from ApoE(-/-) Mice Secrete Biglycan with Hyperelongated Glycosaminoglycan Chains

Atherosclerosis is the major underlying process of cardiovascular disease. Atherosclerosis commences with an initial pre-inflammatory phase of the trapping and accumulation of lipids in the vessel wall and this is followed by an inflammatory response. The trapping of lipids occurs via binding to proteoglycans, specifically biglycan, with hyperelongated glycosaminoglycan (GAG) chains. Models have been developed to study the aetiology as well as the effectiveness of medical and experimental interventions in preventing atherosclerosis. ApoE is an apolipoprotein associated with removal of lipids from peripheral tissues. Disruption of the ApoE gene in C57BL/6 mice produces mice which are ApoE-/- (ApoE deficient) and show elevated plasma lipids and accelerated development of atherosclerosis which is exacerbated on a high fat diet. These mice are widely used in studies of atherosclerosis. We investigated the possibility that changes in the size of biglycan might participate in the development of atherosclerosis in ApoE -/mice. We prepared Aortic Smooth Muscle Cell (ASMC) cultures by the digestion technique from ApoE-/- and ApoE+/+ mice and studied the size of biglycan secreted by both cell types. The biglycan secreted by ASMCs for ApoE -/mice was larger than that from ApoE+/+ ASMCs. The difference was not eliminated by treatment of cells with a high concentration of Platelet Derived Growth Factor (PDGF) or by treatment with the PDGF antagonist, imatinib. The size difference was however observed in the small free GAG chains (xyloside GAGs) secreted in cells supplemented with exogenous xyloside as a cellular assay of GAG synthesizing capacity. This result suggests that there is a hyperactivity of the fundamental GAG synthesizing capacity in the ASMCs from ApoE -/mice. These data suggest that hyperelongated biglycan might be contributing to lipid accumulation in ApoE-/- mice used in studies of atherosclerosis and that some of the medical interventions might have an action to reverse this hyperelongation response.

Thrombin Inhibition Prevents Against Severe Atherosclerosis Progression in Prothrombotic Mice

AbstractAbstract 103 Background:Besides its well-known role in initiation and propagation of thrombus formation, thrombin may have multiple pro-atherogenic actions. Hypothesis:Pharmacologic thrombin inhibition attenuates the onset and progression of atherosclerosis and favors plaque stability in hypercoagulable mice prone to aggressive atherosclerosis development. Methods and Results:Hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerotic burden formation compared to ApoE−/− mice (389.1*103 μm2 vs. 187.0*103 μm2, p<0.0005). Pro-coagulant mice showed larger necrotic cores, decreased plaque collagen content, vast stenosis (up to 90%) and enhanced outward remodeling. Hypercoagulability markedly increased systemic and vascular inflammation, represented by significantly higher CXCL1, MCP-1 and G-CSF plasma levels, pronounced neutrophilia, enhanced Ly6G+ cells arrest to carotid plaques, increased neutrophil recruitment (IHC/Intravital Microscopy), massive intraplaque hemorrhage and spontaneous plaque ruptures. Genetically diminished prothrombin levels (FII-/WT:ApoE−/− mice) caused a significant decrease in atherosclerosis (72.5*103 μm2 vs. 128.4*103 μm2, p<0.01) with a pronounced stable phenotype with less/smaller necrotic cores, thicker fibrous caps and abundant collagen and vascular smooth muscle cell content. Ly6G+ cell intraplaque recruitment was reduced and white blood cell counts and IL-6 levels (3.8±1.66 vs. 8.8±5.34 pg/mL, p<0.05) were diminished compared to control mice. Remarkably, administration of the oral direct thrombin inhibitor Dabigatran etexilate or the anticoagulant activated protein C (APC) in TMPro/Pro:ApoE−/− mice reduced lesions formation by > 80%, suppressed leukocyte recruitment, enhanced plaque stability, whereas numerous pro-inflammatory cytokines were diminished to levels comparable to non-atherosclerotic mice. Conclusion:These data establish a pivotal role for thrombin generation in controlling the level of inflammation related to atherosclerosis development in ApoE−/− mice. The potential clinical impact of long term administration of novel classes of anticoagulants on atherosclerosis merits further study. Disclosures:Weiler:BloodCenter of Wisconsin: Patents & Royalties. van Ryn:Boehringer Ingelheim Pharma GmbH & Co KG: Employment.