| BackgroundRecent study suggests adipose is an active endocrine organ, and adiponectin (APN) is one of its adipocytokines. Now, adiponectin is found to be the most important protective adipocytokine in the body. Low adiponectin level has been found to play an important role in occurrence and development of diabetes, metabolic syndrome and atherosclerosis (AS). Adiponectin can reduce the size of atherosclerotic lesions, improve endothelial cell functions, inhibit neointimal thickening and proliferation of vascular smooth muscle cells, reduce matrix degradation and stable plaques. Through these functions it suppresses atherosclerosis. Therefore, deeply studying the effects and mechanism of adiponectin has important theoretical and clinical significance to prevent and cure atherosclerosis.It has been well known that oxidative stress is one of the major mechanisms involved in atherosclerosis. Reactive oxygen species (ROS) produced by oxidative stress combine with plenty of nitric oxide (NO) in inflammation and produce peroxynitrite (ONOO") quickly. ONOO- initializes nitrative stress and injures protein structure and enzymatic activity, which results in cell apoptosis. Amounts of studies have shown that lipoprotein over oxidation and endothelium injury is the first step in atherosclerosis. And ONOO- produced by nitrative stress is the main substance to overoxidate lipoprotein and injure endothelial cells.Currently, national and international researches about adiponectin mostly focus on its function of improving vascular endothelium via endocrine mode. There are mounts of adipose tissue around vascular adventitia. If adiponectin secreted by these adipose tissues can directly affect plaques in neighboring arteries is still unknown. Our previous studies have suggested adiponectin receptors exist in vascular adventitial fibroblasts and Ad-APN transfer via intima or adventitia can both minish plaque sizes in atherosclerotic rabbits, accompanying with serum adiponectin increase. This study was therefore designed to observe the effects of adiponectin on carotid atherosclerosis plaque and nitrative stress via Ad-APN transfer in adventitia in ApoE-/- mice and explore the possible mechanism.Objectives1. To observe the effects of adiponectin on suppressing carotid atherosclerosis in ApoE-/- mice.2. To study the effect and mechanism of adiponectin on nitrative stress.3. To observe the change of shear stress at plaques in proximate area of collar and explore other possible mechanism of anti-atherosclerosis.Methods1. Establishment of the animal model:50 male ApoE-/- mice,8 weeks of age, were fed on a diet of 0.25% cholesterol and 15% fat for 3 weeks. Then carotid atherosclerosis lesions were induced by perivascular constrictive collar placement on the right common carotid arteries.5 weeks after surgery, mice were randomly divided into 3 groups:control group (n=15), Ad-βgal group (n=15) and Ad-APN group (n=20), which were transfected saline, Ad-βgal and Ad-APN via adventitia, respectively.2 weeks after transfection, all groups were sacrificed by over-dose anesthesia, bloods and tissues were preserved and processed for further assays.2. Ultrasound examinations:At the beginning of the experiment, the end of 8th week and 10th week, maximum plaque thickness, maximum plaque areas, peak velocity (Vp), systolic diameter (Ds), shear stress (SS) were measured by animal ultrasound examinations.3. Virus transfection efficiency examinations:5 days after transfection, GFP of carotid artery sections were observed by fluorescence microscope. 4. Western blot: To examine the protein expressions of adiponectin in the carotid atherosclerosis lesions.5. Histopathology:The carotid artery were processed and examined by HE stain, red oil O stain, Masson stain and immunohistochemical stain of macrophage.6. ELISA assay:To detect the level of nitrotyrosine in atherosclerosis lesions.7. Chemical coloration assay:To detect the level of NO in atherosclerosis lesions.8. Fluorescence stain:To detect the level of O2- in atherosclerosis lesions.9. Statistic analysis:Continuous variables are reported as mean±standard deviation and data was analyzed by SPSS 16.0. For all analysis, a p value less than 0.05 is considered significant.Results1. Establishment of the animal model:One mouse in Ad-P gal group and one in Ad-APN group died after transfection, and other animals finished the experiments. The body weights of all mice were increased significantly at the end of 8th week vs baseline (P<0.01). There were no significant differences among groups at the end of 10th week and between 8th week and 10th week in body weight (P>0.05)2. Virus transfection efficiency:In Ad-βgal group and Ad-APN group, GFP showed green fluorescence obviously in carotid artery plaques and almost no fluorescence in myocardium, liver, and kidney tissues.3. Western blot: Adiponectin expression in plaques was significantly increased in Ad-APN group vs control and Ad-P gal group (P＜0.01)4. Ultrasound examinations:Compared with control group and Ad-βgal group, both maximum plaque thickness and maximum plaque area were significantly reduced in Ad-APN group (P<0.01)Compared with baseline, Vp and SS were decreased significantly at the end of 8th week (P＜0.01). Ds also decreased, but couldn't reach statistic significance (P>0.05). Compared with 8th week, Vp, Ds and SS had no significant changes in every group of 10th week. At the end of 10th week compared with control group and Ad-βgal group, Vp and SS were decreased in Ad-APN group, but couldn't reach statistic significance (P>0.05). Ds in Ad-APN group was increased(P<0.05) and significantly increased (P＜0.01) compared with control group and Ad-βgal group, respectively.5. Histopathology:Compared with control group and Ad-βgal group, plaque sizes of Ad-APN group were obviously decreased, lipid content was reduced, smooth muscle cells and macrophages increased and collagen fiber decreased.6. ELISA assay: Compared with control group and Ad-βgal group, nitrotyrosine level in plaques was decreased in Ad-APN group (P<0.05)7. Chemical coloration assay:Compared with control group and Ad-βgal group, NO concentration in plaques was decreased significantly in Ad-APN group (P＜0.05).8. Fluorescence stain: Compared with control group and Ad-βgal group, red fluorescence (O2-) in plaques was obviously weakened in Ad-APN group.Conclusions1. Ultrasound examinations and histopathology stain both illustrate that adiponectin reduces plaque size and decrease lipid content and the number of macrophages. It proves that adiponectin can suppress atherosclerosis though vascular adventitia interference.2. After adiponectin transfection, the level of nitrotyrosine, NO and O2- all decrease in plaques. It suggests that adiponectin can decrease nitrative stress which is closely connected with oxidative stress and pathological NO production.3. Adiponectin has no significant influence on blood flow velocity and shear stress at plaques in neighboring artery. Its anti-atherosclerosis mechanisms seem to have no relationship with shear stress. |
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