Atherosclerosis In ApoE-deficient Mice Research Articles

Effects of Ethyl Pyruvate in Preventing the Development of Diet-induced Atherosclerosis by Blocking the HMGB1 Expression in ApoE-Deficient Mice.

Ethyl pyruvate (EP) is a lipid derivative of pyruvate and known as an anti-inflammatory agent effective to inhibit many diseases in experimental models. To test the hypothesis that Ethyl pyruvate might prevent atherosclerosis development by blocking the high-mobility group box-1 (HMGB1) expression, 8-week-old ApoE-deficient (ApoE-/-) mice were fed a high-fat diet and treated with EP (50 mg/Kg) or Physiological saline for 12 weeks. THP-1 cells were then differentiated into macrophages by treated with Phorbol-12-myristate-13-acetate (PMA, 100 ng/mL) and incubated with oxidized low-density lipoprotein (oxLDL) only or added with EP (5 mM) for 24 hour. In ApoE-/- mice, EP markedly reduced aortic sinus atherosclerosis lesions with no influence to serum lipid levels. Inhibited HMGB1 expression, decreased macrophages content and reduced Toll-like-receptor2 (TLR2), TLR4, monocyte chemotactic protein 1 (MCP-1) and Tumor Necrosis Factor α (TNF-α) mRNA levels were also observed at the same time. In vitro, EP decreased oxLDL uptake in macrophages and inhibited HMGB1 expression induced by oxLDL. In conclusion, our study indicated that EP was an effective agent against the development of diet-induced atherosclerosis in ApoE-deficient mice by way of blocking the HMGB1 expression.

Dietary Cocoa Powder Improves Hyperlipidemia and Reduces Atherosclerosis in apoE Deficient Mice through the Inhibition of Hepatic Endoplasmic Reticulum Stress.

Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress.

Therapeutic potential of a synthetic FABP4 inhibitor 8g on atherosclerosis in ApoE-deficient mice: the inhibition of lipid accumulation and inflammation

We discovered a synthetic FABP4 inhibitor that ameliorated the symptoms of atherosclerosis and suppressed lipid accumulation.

Suppression of proatherogenic leukocyte interactions by MCS-18 – Impact on advanced atherosclerosis in ApoE-deficient mice

ObjectiveAtherosclerosis is associated with chronic inflammatory responses of the arterial blood vessels. The previously observed protective effect of the MCS-18 substance against the initiation of atherosclerosis in a murine model was explained by its pronounced anti-inflammatory activity. Here, we investigated its impact on murine plaque progression in advanced atherosclerosis and on proatherogenic processes. Approach & ResultsApoE-deficient mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, followed by normal chow and intraperitoneal injections of either MCS-18 (500 μg, n = 10) or saline (n = 10) twice a week for another 12 weeks. Plaque size was reduced in MCS-18 treated mice compared to controls (p = 0.001), which was associated with a reduced size of the lipid core (p = 0.01). There was a decrease in apoptotic cells (p = 0.02), endothelial ICAM-1 expression (p < 0.001), and macrophage density (p = 0.01) in the MCS-18 group. In addition, human and murine dendritic cells (DCs) and human umbilical vein endothelial cells (HUVECs) were treated with MCS-18 (50–200 μg/ml) to analyze cell migration and adhesion under flow conditions. MCS-18 reduced human (p = 0.01) and murine (p = 0.006) DC migration. Furthermore, adhesion of MCS-18-treated DCs to a HUVEC monolayer was decreased (p < 0.001). Compared to controls, CD209 (p < 0.001) and CCR7 (p = 0.003) expression was decreased in MCS-18-treated DCs, while in HUVECs lower levels of ICAM-1 (p < 0.001) and of phosphorylated NF-κB-p65 (p = 0.002) were observed. Blocking of ICAM-1 reduced DC adhesion (p < 0.001). ConclusionsMCS-18 exhibits interesting therapeutic effects when applied in advanced murine atherosclerosis. Its antiatherogenic impact might be associated with a suppressed adhesion to the endothelium due to down-regulation of endothelial ICAM-1 expression.

Abstract 9970: Androgen Deprivation Therapy by Surgical Castration, Gonadotropin Releasing Hormone Receptor Agonists or Antagonists and the Development of Atherosclerosis in Apoe-deficient Mice

Introduction: Adjuvant treatment of prostate cancer often involves androgen deprivation therapy (ADT) by orchiectomy (Orx), gonadotropin releasing hormone (GnRH) receptor agonists or GnRH receptor antagonists. Clinical studies of men with preexisting atherosclerotic disease, have indicated that cardiovascular event rates differs between different types of ADT, suggesting that not only testosterone deficiency, but also direct effects of ADT on processes in the atherosclerotic plaque may be involved. Immune cell GnRH receptors have been implicated as potential mediators. In the present study, we compared the effects of common types of ADT on de-novo atherosclerosis in mice. Methods: Chow-fed Apoe-deficient mice (n=121) were allocated to Orx and/or monthly injections with the GnRH receptor agonist leuprolide (0.5 mg s.c.), the GnRH receptor antagonist degarelix (0.5 mg s.c.) or saline starting from 8 weeks of age (n=19-21 in each group). At 26 weeks of age, the burden of atherosclerosis was quantified by en face analysis of the Oil red O-stained aortic arch and in histological sections of the aortic root. Bone marrow-derived macrophages and splenocytes were incubated in the presence of leuprolide (10 -6 mol/L) or degarelix (10 -6 mol/L). Data is shown as mean ± sem, *p&lt;0.05, **p&lt;0.01. Results: All types of ADT augmented hypercholesterolemia and atherosclerosis with no significant differences in the size of the effect (Figure A-B). In Orx mice, the addition of leuprolide, but not degarelix, led to further increases in plasma total cholesterol and in atherosclerosis in the aortic root (D), but not the aortic arch (C). Plaque necrosis was unaffected by ADT. No effects of the drugs on macrophage cytokine secretion or splenocyte proliferation were observed. Conclusions: ADT did not exert differential effects on the development of atherosclerosis in the Apoe-deficient mice. A pro-atherogenic, possibly cholesterol-mediated, effect of leuprolide was seen in Orx mice.

Co-treatment of Pitavastatin and Dexamethasone Exacerbates the High-fat Diet-induced Atherosclerosis in apoE-deficient Mice.

Activation of macrophage adipocyte fatty acid-binding protein (FABP4) induces development of atherosclerosis in animal models. We previously reported that statin inhibited while dexamethasone activated macrophage FABP4 expression. However, co-treatment of macrophages with statin and dexamethasone induced FABP4 expression in a synergistic manner, which implies that this co-treatment may exacerbate high-fat diet (HFD)-induced atherosclerosis. In this study, we fed apoE-deficient (apoE) mice with HFD or HFD containing dexamethasone or pitavastatin or both for 16 weeks. Compared with HFD alone, pitavastatin or dexamethasone had little effect on lesions in both en face aortas and aortic root cross sections. However, the co-treatment exacerbated HFD-induced lesions. In addition, the co-treatment decreased collagen content and disturbed the integrity of lesion caps. Both serum total cholesterol and LDL cholesterol levels were reduced by pitavastatin and increased by dexamethasone, respectively. However, the co-treatment had little effect on both total cholesterol and LDL cholesterol levels, indicating that the exacerbation of lesions is independent of total cholesterol or LDL cholesterol levels. FABP4 expression in aortic lesion area was significantly induced by the co-treatment, suggesting that activation of FABP4 expression is a main contributor to lesions. In conclusion, our study demonstrates that co-treatment of pitavastatin and dexamethasone exacerbates HFD-induced atherosclerosis and defines a potential risk to use the dual treatment for patients in clinics.

Clopidogrel significantly lowers the development of atherosclerosis in ApoE-deficient mice in vivo.

The anti-platelet drug clopidogrel has been shown to modulate adhesion molecule and cytokine expression, both playing an important role in the pathogenesis of atherosclerosis. The aim of this study was to investigate the impact of clopidogrel on the development and progression of atherosclerosis. ApoE(-/-) mice fed an atherogenic diet (cholesterol: 1%) for 6months received a daily dose of clopidogrel (1mg/kg) by i.p. injection. Anti-platelet treatment was started immediately in one experimental group, and in another group clopidogrel was started 2month after beginning of the atherogenic diet. Blood was analysed at days 30, 60 and 120 to monitor the lipid profile. After 6months the aortic arch and brachiocephalic artery were analysed by Sudan IV staining for plaque size and by morphometry for luminal occlusion. Serum levels of various adhesion molecules were investigated by ELISA and the cellular infiltrate was analysed by immunofluorescence. After daily treatment with 1mg/kg clopidogrel mice showed a significant reduction of atherosclerotic lesions in the thoracic aorta and within cross sections of the aortic arch [plaque formation 55.2% (clopidogrel/start) vs. 76.5% (untreated control) n=8, P<0.05]. After treatment with clopidogrel P-/E-selectin levels and cytokine levels of MCP-1 and PDGFβ were significantly reduced as compared to controls. The cellular infiltrate showed significantly reduced macrophage and T-cell infiltration in clopidogrel-treated animals. These results show that clopidogrel can effectively delay the development and progression of 'de-novo' atherosclerosis. However, once atherosclerotic lesions were already present, anti-platelet treatment alone did not result in reverse remodelling of these lesions.

The enhanced expression of IL-17-secreting T cells during the early progression of atherosclerosis in ApoE-deficient mice fed on a western-type diet.

Atherosclerosis is a chronic progressive inflammatory disorder and the leading cause of cardiovascular mortality. Here we assessed the dynamic changes of T-cell-derived cytokines, such as inteferon (IFN)-γ, interleukin (IL)-17 and IL-4, during the progression of atherosclerosis in apolipoprotein E-null (ApoE−/−) mice, to understand the role of immune responses in different stages of atherosclerosis. Male ApoE−/− mice were fed a high-fat, western-type diet (WD: 21% lipid, 1.5% cholesterol) after 5 weeks of age and were compared with C57BL/6 wild-type control mice fed a standard chow diet. Atherosclerotic lesions appeared in the aortic sinus of ApoE−/− mice 4 weeks after WD and the lesions progressed and occupied >50% of the total sinus area 16 weeks after WD. Aortic IL-17 mRNA and protein expression started to increase in ApoE−/− mice after 4 weeks on the WD and peaked at around 8–12 weeks on the WD. In terms of systemic expression of T-cell-derived cytokines, IL-17 production from splenocytes after anti-CD3/CD28 stimuli increased from 4 weeks on the WD, peaked at 12 weeks and returned to control levels at 16 weeks. The production of IFN-γ and IL-4 (Th1 and Th2 cytokines, respectively) from splenocytes was delayed compared with IL-17. Taken together, the present data indicate that Th17 cell response may be involved at an early stage in the development of atherosclerosis.

Loss of Apoptosis Regulator through Modulating IAP Expression (ARIA) Protects Blood Vessels from Atherosclerosis

Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases.

A single infection with Chlamydia pneumoniae is sufficient to exacerbate atherosclerosis in ApoE deficient mice

Several studies have demonstrated a strong link between Chlamydia pneumoniae (Cp) infection and atherosclerosis progression/exacerbation. Here, we try to understand whether a single administration of Cp could exacerbate atherosclerosis. Apoe−/− mice were intranasally infected with Cp followed by a high fat diet. Mice were sacrificed at different time points after Cp infection to monitor the development of the atheroma. Cp infection increased lipid content in the aortic sinus of Apoe−/− mice starting from 8weeks. This was associated with increased numbers of active myeloid dendritic cells and plasmacytoid DCs which were co-localized with T-cells in the atherosclerotic plaque. The serum levels of IFN-γ showed a Th1-like environment typical of atherosclerosis. In conclusion, we demonstrate that one dose of Cp could exacerbate atherosclerotic lesion development, triggering innate immune cell accumulation early on that allowed the involvement of Th1-like cells in the exacerbation of the atherosclerotic plaque at later time points.

Clopidogrel Significantly Lowers the Development of Atherosclerosis in ApoE-Deficient Mice in vivo

The anti-platelet drug clopidogrel has been shown to modulate adhesion molecule and cytokine expression, both playing an important role in the pathogenesis of atherosclerosis. The aim of this study was to investigate the impact of clopidogrel on the development and progression of atherosclerosis. ApoE−/− mice fed an atherogenic diet (cholesterol: 1 %) for 6 months received a daily dose of clopidogrel (1 mg/kg) by i.p. injection. Anti-platelet treatment was started immediately in one experimental group, and in another group clopidogrel was started 2 month after beginning of the atherogenic diet. Blood was analysed at days 30, 60 and 120 to monitor the lipid profile. After 6 months the aortic arch and brachiocephalic artery were analysed by Sudan IV staining for plaque size and by morphometry for luminal occlusion. Serum levels of various adhesion molecules were investigated by ELISA and the cellular infiltrate was analysed by immunofluorescence. After daily treatment with 1 mg/kg clopidogrel mice showed a significant reduction of atherosclerotic lesions in the thoracic aorta and within cross sections of the aortic arch [plaque formation 55.2 % (clopidogrel/start) vs. 76.5 % (untreated control) n = 8, P < 0.05]. After treatment with clopidogrel P-/E-selectin levels and cytokine levels of MCP-1 and PDGFβ were significantly reduced as compared to controls. The cellular infiltrate showed significantly reduced macrophage and T-cell infiltration in clopidogrel-treated animals. These results show that clopidogrel can effectively delay the development and progression of ‘de-novo’ atherosclerosis. However, once atherosclerotic lesions were already present, anti-platelet treatment alone did not result in reverse remodelling of these lesions.

Effect of Repetitive Glucose Spike and Hypoglycaemia on Atherosclerosis and Death Rate in Apo E-Deficient Mice.

Epidemiological data suggest that postprandial hyperglycaemia and hypoglycaemia are potential risk factors for cardiovascular disease. However, the effects of repetitive postprandial glucose spikes, repetitive hypoglycaemia, and their combination on the progression of atherosclerosis remain largely unknown. The present study investigated the effects of rapid rises and falls in glucose, and their combination, on the progression of atherosclerosis in apolipoprotein (apo) E-deficient mice. In this study, apo E-deficient mice with forced oral administration of glucose twice daily for 15 weeks were used as a model of repetitive postprandial glucose spikes, and apo E-deficient mice given an intraperitoneal injection of insulin once a week for 15 weeks were used as a model of repetitive hypoglycaemia. In addition, we established a model of both repetitive postprandial glucose spikes and hypoglycaemia by combining the above interventions. Atherosclerosis was evaluated in all mice by oil red O staining. Administration of ipragliflozin, a selective inhibitor of sodium-glucose cotransporter 2, in the mouse model of repetitive glucose spikes inhibited the progression of atherosclerosis, whereas long-term repetitive glucose spikes, repetitive hypoglycaemia, and their combination had no significant impact on atherosclerosis. However, repetitive hypoglycaemia was associated with poor survival rate. The results showed that repetitive hypoglycaemia reduces the survival rate without associated progression of atherosclerosis in apo E-deficient mice.

Bu-shen-he-mai-fang (HMF) Decoction Inhibits Atherosclerosis by Improving Antioxidant and Anti-Inflammatory Activities in ApoE-deficient Mice

Objective: To observe the effects of Bu–shen- he- mai- fang (HMF) on experimental atherosclerosis in ApoE-deficient mice. Materials an Methods: Thirty male ApoE-deficient mice were randomly divided into 3 groups (10 mice per group) as follows: one group received the standard high- cholesterol diet (high- cholesterol group, HC); Another group received high- cholesterol diet supplemented with HMF decoction 1.37 g/kg/day; the third group received a high- cholesterol diet, supplemented with atrovastatin 5 mg/kg/day for 8 weeks. The extent of atherosclerosis, the expression of LOX-1 protein and macrophage infiltration were evaluated by H&amp;E, oil red O staining, and immunohistochemical staining. SOD was also measured by a spectrophotometer. Results: The degree of atherosclerosis was significantly lower in HMF group and atrovastatin group than that in high-cholesterol group. The expression of LOX-1 protein and macrophage filtration were significantly lower in HMF group and atrovastatin group than that in high-cholesterol group. Also, the SOD was higher in HMF group and atrovastatin group than that in high-cholesterol group. Conclusion: The results suggested that HMF significantly inhibited early atherosclerotic lesions by inhibiting inflammatory response and decreasing the generation of ROS.

Anti-atherogenic effect of hydrogen sulfide by over-expression of cystathionine gamma-lyase (CSE) gene.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule that functions in physiological and pathological conditions, such as atherosclerosis. H2S dilates vessels and therefore has been suggested as an anti-atherogenic molecule. Since cystathionine gamma-lyase (CSE) enzyme is responsible for producing H2S in the cardiovascular system, we hypothesized that up-regulation of CSE expression in vivo with preservation of H2S bioactivity can slow down plaque formation and, can serve as a therapeutic strategy against atherosclerosis. In this study, C57BL/6 wild type mice (WT), ApoE knockout mice (KO) and transgenic ApoE knockout mice overexpressing CSE (Tg/KO) at four weeks of age were weaned. They were then fed with either normal or atherogenic diet for 12 weeks. At week 16, serial plasma lipid levels, body weight, and blood pressure were measured prior to euthanization of the mice and the size of atherosclerotic plaques at their aortic roots was measured. Tg/KO mice showed an increase in endogenous H2S production in aortic tissue, reduced atherosclerotic plaque sizes and attenuation in plasma lipid profiles. We also showed an up-regulation in plasma glutathionine peroxidase that could indicate reduced oxidative stress. Furthermore, there was an increase in expression of p-p53 and down regulation of inflammatory nuclear factor-kappa B (NF-κB) in aorta. To conclude, alteration of endogenous H2S by CSE gene activation was associated with reduced atherosclerosis in ApoE-deficient mice. Up-regulation of CSE/H2S pathway attenuates atherosclerosis and this would be a potential target for therapeutic intervention against its formation.

The thyroid receptor β modulator GC-1 reduces atherosclerosis in ApoE deficient mice

Thyroid hormone reduces plasma cholesterol and increases expression of low-density lipoprotein receptor (LDL-R) in liver, an effect mediated by thyroid receptor β (TRβ). The selective TRβ modulator GC-1 also enhances several steps in reverse cholesterol transport and can decrease serum cholesterol independently of LDL-R. To test whether GC-1 reduces atherosclerosis and to determine which mechanisms are active, we treated ApoE deficient mice with atherogenic diet ± GC-1. GC-1 reduced cholesteryl esters in aorta after 20 weeks. Serum free and esterified cholesterol were reduced after 1 and 10 weeks, but not 20 weeks. Hepatic bile acid synthesis and LDL-R expression was elevated after 1, 10 and 20 weeks, without changes in hepatic de novo cholesterol synthesis. GC-1 increased faecal neutral sterols and reduced serum campesterol after 1 week, indicating reduced intestinal cholesterol absorption. After 20 weeks, GC-1 increased faecal bile acids, but not faecal neutral sterols. Hepatic scavenger receptor B1 (SR-B1) expression was decreased by GC-1. We conclude that GC-1 delays the onset of atherosclerosis in ApoE deficient mice. Since ApoE is needed for hepatic cholesterol reabsorption by LDL-R, this supports the idea that GC-1 reduces serum cholesterol independently of LDL-R by increasing hepatic bile acid synthesis. GC-1 lipid-lowering effects in ApoE deficient mice may also be partly due to reduced intestinal cholesterol absorption. Since reductions in serum cholesterol are reversed at longer times, these GC-1 dependent effects may not be enough for sustained cholesterol reduction in long term treatments.

128: Modulation of atherosclerosis in ApoE-deficient mice by gp130 signalling

Interleukin (IL)-6 is a key modulator of the acute phase response (APR), and while both have been implicated in atherosclerosis, the pathological role of signalling cascades downstream of IL-6 is ill-defined. Since IL-6 employs the cytokine receptor gp130 to primarily activate the STAT3 signalling cascade, here we evaluate whether gp130-dependent STAT3 activation modulates atherosclerosis. High-fat diet-induced atherosclerosis was established in ApoE −/− mice crossed with gp 130 F / F knock-in mice displaying elevated gp130-dependent STAT3 activation and production of the APR protein, serum amyloid A (SAA). Also generated were gp 130 F / F :Stat3 −/+ :ApoE −/− mice displaying genetically-normalised STAT3 activation and SAA levels, and bone marrow chimeras involving ApoE −/− and gp 130 F / F :ApoE −/− mice. At 10 weeks post high-fat diet, aortic atherosclerotic lesions, including the presence of CD68 + macrophages, and plasma lipid and SAA profiles, were assessed. Aortic plaque development and plasma triglyceride levels in gp 130 F / F :ApoE −/− mice were significantly reduced (3-fold, P < 0.001) compared to ApoE −/− littermates. By contrast, in gp 130 F / F :ApoE −/− mice, atherosclerotic plaques contained augmented CD68 + macrophage infiltrates, and plasma SAA levels were elevated, compared to ApoE −/− mice. Atherosclerotic lesion development and plasma triglyceride levels in gp 130 F / F :ApoE −/− and gp 130 F / F :Stat3 −/+ :ApoE −/− mice were comparable, despite a significant ( P < 0.05) reduction in macrophage numbers in lesions, and also plasma SAA levels, in gp 130 F / F :Stat3 −/+ :ApoE −/− mice. Finally, aortic plaque development and plasma triglyceride levels were comparable in ApoE −/− mice reconstituted with gp 130 F / F :ApoE −/− (ApoE F / F :ApoE) or ApoE −/− (ApoEApoE) bone-marrow cells. Deregulation of gp130/STAT3 signalling augments the APR and macrophage infiltration during atherosclerosis without impacting on the development of aortic plaques.

60: Atherosclerosis severity is independent of endogenous IL-33 signaling

Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work demonstrated that the systemic administration of IL-33 reduces the development of atherosclerosis in the apolipoprotein E-deficient (ApoE −/− ) mouse model of the disease by induction of a Th1-to-Th2 shift. However, the role of endogenous IL-33 in the atherogenesis remains elusive. Atherosclerosis was induced in 10 week-old ApoE −/− , IL-33 −/− ApoE −/− and ST2 −/− ApoE −/− mice by feeding a high-cholesterol diet (1.25%, no cholate) for 10 weeks. Additionally, a group of ApoE −/− mice were injected with a neutralizing anti-ST2 antibody or an isotype control during the period of the diet. The atherosclerotic lesion development was measured with Oil Red O in the thoracic-abdominal aorta and in the aortic sinus. The mRNA levels of several cytokines, including IL-6, IFN γ , IL-17, IL-5 and IL-10 were assessed in the aorta and in in vitro -stimulated lymph node cells. We observed no differences in lipid-staining area in the aortas of IL-33 −/− ApoE −/− mice (8.84 ± 0.97; mean ± SEM; n = 9–25), ST2 −/− ApoE −/− mice (6.95 ± 0.78), ApoE −/− mice untreated (7.05 ± 0.78), ApoE −/− mice injected with either the neutralizing anti-ST2 antibody (6.08 ± 0.79) or the isotype control (6.16 ± 0.86) after high-cholesterol diet feeding. Similar results were obtained in the aortic sinus compared to ApoE −/− controls. Total serum cholesterol and triglyceride levels were not different compared to ApoE −/− controls. IL-33 expression in aortic tissue was comparable in ApoE −/− and ST2 −/− ApoE −/− mice and absent in IL-33 −/− ApoE −/− mice. There was no difference in the transcript levels of inflammatory cytokines in the aorta and in in vitro -stimulated lymph node cells. These data indicate that in contrast to the anti-atherosclerotic effect of systemically administered recombinant IL-33, the endogenously produced cytokine and its receptor do not significantly influence the severity of atherosclerosis in ApoE-deficient mice fed with a high-cholesterol diet.

MCS-18, a natural product isolated from Helleborus purpurascens, inhibits maturation of dendritic cells in ApoE-deficient mice and prevents early atherosclerosis progression

IntroductionInflammation accelerates both plaque progression and instability in the pathogenesis of atherosclerosis. The inhibition of dendritic cell (DC) maturation is a promising approach to suppress excessive inflammatory immune responses and has been shown to be protective in several autoimmune models. The aim of this study was to investigate the immune modulatory effects of the natural substance MCS-18, an inhibitor of DC maturation, regarding the progression of atherosclerosis in ApoE-deficient mice. Materials and methodsApoE-deficient mice were fed for twelve weeks with a Western-type diet (n = 32) or normal chow (control group; n = 16). Animals receiving high-fat diet were treated with MCS-18 (500 μg/kg body weight, n = 16) or saline (n = 16) twice a week. After 12 weeks, animals were transcardially perfused and sacrificed. The percentage of mature DCs (CD3−/CD19−/CD14−/NK1.1−/CD11c+/MHCII+/CD83+/CD86+) and T cell subpopulations (CD4+/CD25+/Foxp3+, CD3/CD4/CD8) was analyzed in peripheral blood and in the spleen using flow cytometry. Plaque size was determined in the aortic root and the thoracoabdominal aorta using en-face staining. Immunohistochemical stainings served to detect inflammatory cells in the aortic root. Several cytokines and chemokines were determined in serum using multiplex assays. ResultsIn splenic cells derived from saline-treated atherosclerotic mice an increased DC maturation, reflected by the upregulation of CD83 and CD86 expression, was observed. The enhanced expression of both maturation markers was absent in MCS-18 treated atherosclerotic mice. While the percentage of splenic Foxp3 expressing Treg was increased in animals receiving MCS-18 compared to saline-treated atherosclerotic mice, cytotoxic T cells were reduced in the spleen and in atherosclerotic lesions of the aortic root. Furthermore, proatherogenic cytokines (e.g. IL-6 and IFN-γ) and chemokines (e.g. MIP-1β) were decreased in serum of MCS-18-treated animals when compared to saline-treated atherosclerotic mice. Also plaque size in the aortic root and the thoracoabdominal aorta was significantly lower following administration of MCS-18. ConclusionThis study provides for the first time evidence that MCS-18 is able to prevent the onset of atherosclerosis in ApoE-deficient mice. The observed anti-atherogenic effect is associated with the suppression of DC maturation and an inhibited migration and proliferation of cytotoxic T cells.

Effect of Lowering Asymmetric Dimethylarginine (ADMA) on Vascular Pathology in Atherosclerotic ApoE-Deficient Mice with Reduced Renal Mass

The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1), on atherosclerosis in subtotally nephrectomized (SNX) ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n = 39) and C57Bl/6J wild-type littermates (WT, n = 27) with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction) ApoE-competent DDAH1 transgenic and wild-type littermates (n = 11) served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD) in this mouse model.

Thrombin inhibition by dabigatran attenuates atherosclerosis in ApoE deficient mice.

IntroductionAtherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now.Material and methodsAtherosclerosis prone 12 weeks old ApoE–/– mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day).ResultsThe thrombin time (HEMOCLOT®) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment.ConclusionsThe thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.