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AbstractAbstract - selectedArticleFigures/Tables Figures/Tables - selectedReferences References - selected   European Journal of Pharmacology
Volume 649, Issues 1-3, 15 December 2010, Pages 285-292

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doi:10.1016/j.ejphar.2010.09.027 | How to Cite or Link Using DOI   Permissions & Reprints

Cardiovascular Pharmacology

WIN55212-2 ameliorates atherosclerosis associated with suppression of pro-inflammatory responses in ApoE-knockout mice

This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevier.

Yan Zhao^a, Yan Liu^a, Weiping Zhang^a, Jiahong Xue^a, Yue Z. Wu^a, Wei Xu^a, Xiao Liang^a, Tao Chen^a, Chiharu Kishimoto^c and Zuyi Yuan^{a, b, ,}

^a Department of Cardiovascular Medicine, the First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi 710061, China

^b Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, Shaanxi 710061, China

^c Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606–8507, Japan

Received 26 January 2010;  revised 27 August 2010;  accepted 15 September 2010.  Available online 21 September 2010.

Abstract

The role of inflammation in all stages of atherosclerosis has been actively investigated, with an emphasis on the discovery of novel and innovative drugs for treatment and prevention. The anti-inflammatory and immunomodulatory capacity of cannabinoids are well established, and these agents have a broad therapeutic potential in various inflammatory diseases, including cardiovascular diseases. The aim of this study was to investigate the effect of WIN55212-2, a synthetic cannabinoid, on atherosclerosis using the apolipoprotein E-knockout (ApoE^−/−) mouse on a cholate-containing high-fat diet. Our results showed that WIN55212-2 reduced the size of atherosclerotic lesions in the aorta root, and did not affect serum lipid levels significantly. Furthermore, alleviation of atherosclerosis by WIN55212-2 was associated with a smaller content of macrophages in plaque lesion as well as decreasing pro-inflammatory gene and NF-κB activation in aortic tissues. Oxidized LDL (ox-LDL) dramatically induced NF-κB activation, and enhanced pro-inflammatory mRNA and protein in peritoneal macrophages isolated from ApoE^−/− mice. It is noteworthy that all of the above-mentioned effects of ox-LDL were attenuated by WIN55212-2. Moreover, WIN55212-2 also attenuated the inflammatory response that LPS induced. AM630, a cannabinoid receptor 2 (CB₂) special antagonist completely abolished the protective effects of WIN55212-2 both in vivo and in vitro. Our data provide strong evidence that WIN55212-2 can potentially inhibit atherosclerosis in ApoE^−/− mice. Importantly, all the beneficial effects of WIN55212-2 in our model were closely associated with the suppression of pro-inflammatory responses and were mediated by the CB₂ receptor.

Keywords: WIN55212-2; Atherosclerosis; Inflammation; Macrophage

Article Outline

1.

Introduction

2.

Materials and methods

2.1. Experimental atherosclerosis

2.2. Drugs and treatment protocols

2.3. Lipid measurements

2.4. Quantification of atherosclerotic lesions by immunohistochemistry

2.5. LDL isolation and oxidation

2.6. Cell culture

2.7. RNA isolation and quantitative RT-PCR

2.8. Protein preparation

2.9. Western blotting

2.10. Measurement of MCP-1, IL-6 and TNF-α concentration

2.11. Electrophoretic mobility-shift assay (EMSA)

2.12. Statistical analysis

3.

Results

3.1. CB₂ receptors are expressed in plaque lesion and peritoneal macrophages

3.2. Effects of WIN55212-2 on physiological parameters

3.3. WIN55212-2 reduces atherosclerotic lesion size in ApoE^−/− mice

3.4. Immunohistochemical analysis of macrophage accumulation in plaque lesion

3.5. WIN55212-2 reduced inflammatory gene in plaque lesions

3.6. WIN55212-2 reduced NF-κB activation in advanced plaque lesions

3.7. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage inflammatory gene

3.8. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage NF-κB activation and IκB phosphorylation

4.

Discussion

Acknowledgements

References

Fig. 1. 

Cannabinoid CB₂ receptors are expressed in aortic tissue of ApoE^−/− mice and in peritoneal macrophages. A) of CB₂ receptors in a plaque lesion of ApoE^−/− mice demonstrated by staining. B) Analysis of CB₂ using Western blotting in aortic tissue and peritoneal macrophages. C) The detection of CB₂ gene using RT-PCR in aortic tissue and peritoneal macrophages.

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Fig. 2. 

WIN55212-2 reduces the development of atherosclerotic lesions in ApoE^−/− mice. Littermate male ApoE^−/− mice were given a daily injection i.p. of WIN55212-2, AM630 plus WIN55212-2, or vehicle alone for 8 weeks starting at 14 weeks old. A) Representative photomicrographs of Oil red O-stained fatty streaks (n = 8). B) and C) Quantitative analysis of atherosclerotic lesion sizes in the aortic root. Magnification, 40×. Data are presented as the mean ± S.D.; n = 8 per group; *P < 0.05.

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Fig. 3. 

Effect of WIN55212-2 on macrophage (Mφ) accumulation in the lesions. Frequency of Mφ in WIN55212-2 mice is decreased dramatically compared with that of the control mice. In contrast, treatment with AM630 plus WIN55212-2 increased the frequency of Mφ. A) Representative photomicrographs of macrophages stained with Mac₃. B) Quantitative analysis of the percentage of lesion area occupied by macrophages. Magnification, 400×. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

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Fig. 4. 

Inhibitory effects of WIN55212-2 on of inflammatory mRNA in the aortic arch isolated from ApoE^−/− mice. The of the inflammatory genes, including IL-6 (A), TNF-α (B) and MCP-1 (C), was measured by real-time RT-PCR. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

View Within Article

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Fig. 5. 

Inhibitory effect of WIN55212-2 on NF-κB activation in aortic arch tissue isolated from ApoE^−/− mice. Nuclear proteins were extracted from aortic arch tissue. NF-κB activation was determined using p65 Western blot as described in Materials and methods. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

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Fig. 6. 

Effect of WIN55212-2 on of inflammatory gene mRNA. IL-6(A), (E), TNF-α(B), (F)and MCP-1(C), (G) mRNA levels were measured by real-time RT-PCR in triplicate. The data are presented as the mean ± S.D.; *P < 0.05 vs. the control, #P < 0.01 vs. the ox-LDL or LPS pre-incubation group.

View Within Article

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Fig. 7. 

Electrophoretic mobility-shift assay of nuclear extracts and p-IκB-α in peritoneal macrophages isolated from ApoE^−/− mice. Isolated peritoneal macrophages were cultured without (control) or with WIN55212-2 or with WIN55212-2 plus AM630 as described in Materials and methods. EMSA experiments with the consensus sequence for NF-κB and p-IκB-α were done as described in Materials and methods.

View Within Article

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Fig. 8. 

Effect of WIN55212-2 on the release of inflammatory cytokines MCP-1, IL-6 and TNF-α in peritoneal macrophage isolated from ApoE^−/− mice. Isolated peritoneal macrophage were cultured without (control) or with WIN55212-2 or with WIN55212-2 plus AM630 as described in Materials and methods. The levels of IL-6 (A), (E), MCP-1(B), (F) and TNF-α (C), (G) were measured by ELISA. Values are presented as mean ± S.D. All results shown are representative of the results of three independent experiments. *P < 0.05 vs. control, # P < 0.05 vs. ox-LDL or LPS pre-incubation group.

View Within Article

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Table 1. Primer sequences.

View Within Article

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Table 2. Physiological parameters in ApoE^−/− mice at sacrifice.

Values are means ± SD; treatment with WIN55212-2 or WIN55212-2 plus AM630 did not significantly modify the physiological parameters of ApoE^−/− mice.

View Within Article

Corresponding author. Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, 277 West Yanta Road, Xi'an, Shaanxi 710061, China. Tel.: + 86 29 8532 3819; fax: + 86 29 8532 3709. Copyright © 2010 Elsevier B.V. All rights reserved.

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European Journal of Pharmacology
Volume 649, Issues 1-3, 15 December 2010, Pages 285-292  

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