Contributed equally
Adiponectin (APN) is an important anti-atherogenic adipocytokine. The aim of the present study was to investigate the role of adiponectin in atherosclerotic plaque formation and clarify its mechanisms. An atherosclerosis model was induced by
All of the clinical and experimental studies on adiponectin (APN) markedly suggest that it is a critical vascular protective molecule and its reduction may contribute to vascular injury in metabolic disorder-associated diseases (
Oxidative stress, comprising the production of ·O2− and its derivative peroxynitrite (ONOO−), contributes to the onset of atherosclerosis (
Therefore, the present study aimed to determine the relevance of adiponectin and its attenuation of oxidative stress. It was identified that adiponectin may reduce atherosclerotic plaque and improve the stability of plaques in ApoE−/− mice with atherosclerosis by inhibiting inducible nitric oxide synthase (iNOS) and oxidative/nitrative stress.
The adenovirus producing the full-length adiponectin was constructed with use of the Adenovirus (Ad) Expression Vector kit (Takara, Kyoto, Japan) as described previously (
Male ApoE−/− mice (10–12 weeks old; Beijing University, Beijing, China) were housed at room temperature (24°C) under a 12-h dark/light cycle and received a high-fat diet (0.25% cholesterol and 15% cocoa butter) for 12 weeks. Carotid atherosclerotic lesions were induced by surgical placement of perivascular constrictive silica collars (Shandog Medical Instrument Institute, Shandong, China) on the left common carotid arteries as described by our group previously (
The Vevo770 ultrasonography system (Visualsonics, Toronto, Canada; 55-MHz scan head, 4.5-mm focus, axial resolution 30 μM) was used to measure the baseline parameters of the left carotid artery at the beginning of the experiment prior to and following transfection as previously described (
Frozen carotid-artery cross-sections (10-μM thick) embedded in optimal cutting temperature compound (OCT; Sakura Finetechnical Co., Ltd., Tokyo, Japan) following overnight fixation in 10% formalin were mounted on slides. Three sections (200-μM apart) for each mouse were stained with Oil-red O (Ameresco, Solon, OH, USA) and Masson trichrome (Maxim-Bio, Fujian, China). The corresponding sections were measured by use of an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, Rockville, MD, USA) attached to a color CCD video camera (BX51; Olympus Corp., Tokyo, Japan).
The carotid tissue was rinsed and homogenized. The content of NO in the supernatant was determined using the Griess Reagent method (Nitric Oxide Assay kit, Beyotime, Shanghai; Siever 280i NO Analyzer) as described previously (
The protein from the carotid tissue homogenates was separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with monoclonal antibodies against adiponectin, iNOS (both Upstate Biotechnology, Inc., Lake Placid, NY, USA) and β-actin (Cell Signaling Technology, Inc., Beverly, MA, USA), then horseradish peroxidase-conjugated anti-mouse immunoglobulin G antibody (1:2,000, Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h. The blots were developed by using a supersignal chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and visualized using a Kodak Image Station 400 (Amersham Pharmacia, Deisenhofen, Germany). The sample loadings were normalized with anti-β-actin polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) and quantification involved use of the Image Station 2000R system (Eastman Kodak, Rochester, NY, USA).
Paraformaldehyde-fixed tissues were cut into semi-thin sections 4–5-μM thick and stained with the following antibodies: mouse anti-macrophage/monocyte monoclonal (clone MOMA-2; 1:500; Millipore, Billerica, MA, USA), mouse anti-human anti-α-actin (1:500; Millipore) and mouse anti-nitrotyrosine (clone 1A6; 1:200; Millipore). Immunohistochemistry staining was developed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Plasma adiponectin levels were determined by use of a mouse adiponectin ELISA kit (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA) according to the manufacturer’s instructions.
The nitrotyrosine content in the carotid tissue, indicating
All data are expressed as the mean ± standard error. Comparisons between the various groups were analyzed by one-way analysis of variance followed by Dunnett’s post-hoc test using SPSS 16.0 software (International Business Machines, Armonk, NY, USA). P<0.05 (two-sided) was considered to indicate a statistically significant difference.
On the 7th day following virus injection, the expression of virus-targeted genes was detectable in the mice, without loss of weight. As demonstrated in
Protein levels of adiponectin in the carotid tissue were further studied. The results demonstrated that protein levels of adiponectin in Ad-APN plaques were higher than those in the Ad-β-gal and PBS plaques (2.5±0.2 vs. 1.3±0.1; P<0.05; 2.5±0.2 vs. 1.0±0.15; P<0.05;
The present study used micro-ultrasonography to evaluate the plaques. Prior to surgery, the carotid intima of control mice was smooth (
Based on the micro-ultrasonography results, the plaque composition was further analyzed (
Oxidative and nitrative stresses are primary factors responsible for atherosclerosis. To further assess the mechanism of altered atherosclerotic lesions in ApoE−/− mice, superoxide and NO production were detected. As summarized in
The aforementioned results suggested that the reduced superoxide production and prevention of NO destruction may contribute to the vasculoprotective effect of APN in ApoE−/− mice. To identify the molecular candidates responsible for this effect, the iNOS protein expression was determined. It was identified that iNOS activity was reduced in the Ad-APN-treated mice (P<0.05;
Numerous studies have demonstrated that adiponectin is an important antiatherogenic adipocytokine that inhibits insulin resistance, inflammation and oxidative stress (
Adiponectin is secreted by adipose tissue and has a significant role in the development of cardiovascular diseases. The incidence of cardiovascular mortality is increased in patients with low plasma adiponectin compared with that with higher plasma adiponectin levels (
Evidence suggests that common risk factors for atherosclerosis increase the risk of free ROS production from endothelial cells and from smooth muscle and adventitial cells (
Oxidative/nitrative stress due to increased iNOS and superoxide production and subsequent cytotoxic ONOO− production are early hallmarks of vascular injury in patients with atherosclerosis. The physiological or pharmacological concentrations of NO exert significant cardioprotective effects, whereas the reaction product of NO and ·O2−, ONOO−, is highly cytotoxic. Several lines of evidence indicated that adiponectin improves endothelial function by its antioxidative/antinitrative properties and by enhancing eNOS activity, blocking iNOS and NADPH oxidase expression and ONOO− production (
In conclusion, these results demonstrated that adiponectin, as a unique cytokine, reduced atherosclerosis and attenuates oxidative/nitrative stress by blocking iNOS, superoxide and ONOO− production. The present study took a different approach and provided evidence that adiponectin expression significantly reduced atherosclerosis associated with oxidative/nitrative stress.
This study was supported by The National Basic Research Program of China (973 Program, 2011CB503906), Development Program of China (863 Program, 2007AA02Z448), the National Natural Science Foundation of China (nos. 30728025 and 30970709), National Natural Science Funds for Young Scholar (no. 81200211), grants from the Shandong Young Scientists Award Fund (no. BS2012SW003) and grants from Scientific and technology Development program of Jinan (nos. 201003125 and 200905035).
APN levels in atherogenic plaque in ApoE−/− mice. Green fluorescence protein expression in atherogenic plaque seven days following injection with (A) PBS, (B) Ad-β-gal and (C) Ad-APN (magnification, ×10). (D) Relative APN levels in atherogenic plaque in PBS, Ad-β-gal and Ad-APN groups. Values are expressed as the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. Ad-β-gal. PBS, treated with PBS; Ad-β-gal, transfected with empty vector; Ad-APN, transfection with Ad-APN. ApoE−/−, apolipoprotein E-deficient; Ad-β-gal, Ad-β-galactosidase; PBS, phosphate-buffered saline; APN, adiponectin; Ad, adenovirus.
Micro-ultrasonographic evaluation of the plaque. (A) The carotid intima of control mice was smooth prior to surgery. (B) Abundant atherosclerotic lesions were visible in the lumen six weeks following surgery. (C–E) Following two weeks of transfection, the atherosclerotic plaques in the left common carotid arteries were observed on micro-ultrasonography in (C) the PBS group, (D) The Ad-β-gal group and (E) the Ad-APN group. PBS, treated with PBS; Ad-β-gal, transfected with empty vector; Ad-APN, transfected with adiponectin adenovirus. Ad-β-gal, Ad-β-galactosidase; PBS, phosphate-buffered saline.
Effect of APN on plaque composition in carotid atherosclerotic lesions in ApoE−/− mice. (A) Representative microscopic images of the types of atherosclerotic plaques (magnification, ×20). The plaque composition was comparable among treatment groups (PBS, Ad-β-gal transfection and Ad-APN transfection) two weeks following transfection. (B–D) Quantification of the immunohistochemical stains. (B) Lipid content assessed by Oil-red O staining; (C) immunohistochemical detection of macrophages; and (D) α-actin, for smooth muscle in atherosclerotic lesions. PBS, transfection with PBS; Ad-β-gal, transfection with empty vector; Ad-APN, transfection with Ad-APN. ApoE−/−, apolipoprotein E-deficient; PBS, phosphate-buffered saline; APN, adiponectin; Ad, adenovirus; VSMC, vascular smooth muscle cell.
Effect of APN on oxidative/nitrative stress in carotid atherosclerotic lesions in ApoE−/− mice. (A) Detection of superoxide content (magnification, ×40). (B) Immunohistochemical detection of nitrotyrosine and nitrotyrosine content in carotid tissues (magnification, ×40). (C) NO content. (D) Western blot analysis of iNOS protein levels. The values are the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. Ad-β-gal. PBS, treated with PBS; Ad-β-gal, transfected with empty vector; Ad-APN, transfected with Ad-APN. iNOS, inducible nitric oxide synthase; ApoE−/−, apolipoprotein E-deficient; PBS, phosphate-buffered saline; APN, adiponectin; Ad, adenovirus.