Relationship Between Adiponectin And Intimal Proliferation After Balloon Denudation Of Iliac Artery In Rabbits And The Effects Of Rosiglitazone

BackgroundsPTCA(percutaneous transluminal coronary angioplasty) is an ideal method of the treatment with coronary artery stenosis disease in recent years,but the high incidence of postoperative restenosis seriously affect its long-term efficacy,the patients and society has been caused enormous economic burden.The mechanism of the restenosis is still unknown yet.Dysregulation of adipocyte-derived bioactive molecules plays an important role in the development of atherosclerosis and restenosis.Adiponectin is an adipocyte-specific plasma protein discovered recently,a lot of research has been shown that adiponectin have effects of anti-atherogenic, anti-oxidation,anti-inflammatory,insulin sensitization effect,etc,but its role in preventing restenosis after PTCA is not clear yet.The expression of adiponectin in vivo was regulated by many factors,TZDs(thiazolidinedions) drugs is comparatively a typical one,It can increase the expression of adiponectin levels in vivo through the excitement of the PPAR(peroxisome proliferator activated receptor) gamma.Peroxisome proliferator-activated receptor-γhas been shown to be expressed in many of the cells that play important role in the response to vascular injury.As one of insulin sensitizing agents,TZD drugs can be activated after combine with its receptor, and have a direct impact on the diabetic patients' insulin resistance,hyperinsulinemia and hyperglycemia metabolic disorders,at the same time,these drugs also showed a good effects in lowering blood pressure,regulating lipid metabolism,inhibiting inflammatory response,anti-atherosclerosis,and the protection of the kidney.But the effect of TZD drugs on restenosis after PTCA is rare reported yet.Our research on it will help clarify its link with fat factors like adiponectin,and will be useful of our clinical work,give new ideas to solve this problem of restenosis,And will provides new evidence for the link of diabetes,fat metabolism disorders and cardiovascular disease.Objective1.Investigate the relationship between adiponectin and intimal proliferation after balloon endothelial denudation,and do correlation analysis in order to definite the effect of APN in vascular restenosis after balloon injury.2.To analysis the effects of specificity PPARγactivator rosiglitazone on neointimal formation,proliferation and apoptosis of smooth muscle cells,and adiponectin level of hypercholesterolemic rabbits following vascular injury.Explore the mechanism of rosiglitazone in prevention restenosis.3.Investigate the effect of rosiglitazone on blood glucose,lipid and C-reactive protein after hypercholesterolemic and balloon injury.Methods1.Animals and dietThirty three-month-old male New Zealand rabbits,weighing 2.8±3.5 Kg,were utilized for this experiment.Animals were handled in compliance with the Guiding Principles in the Care and Use of Animals.after one week of the feeding adaptation, thirty rabbits were randomly divided into three groups:operation control group (normal diet plus ligatured of femoral artery),model group(hypercholesterol diet plus balloon endothelial denudation) and rosiglitazone groups(balloon endothelial denudation plus hypercholesterol diet supplemented with rosiglitazone),each groups have ten rabbits.During first eight weeks the animals were fed a hypercholesterole diet(1%cholesterol,8%lard and 7.5%dried egg yolk added to the standard diet ). Normal groupe was only fed with standard diet.Subsequently,The right iliac artery in rabbits were injured by balloon in model group and therapy group.2.Construction of vascular injury modelThe rabbits underwent balloon catheter(2.5F/3 atm/5 min) injury of the right iliac artery through the femoral artery on the eight weeks of the experiment.After conventional disinfection,The subcutaneous tissue was separated,exposed femoral artery 2-3 cm,The farend of femoral artery was ligatured,and he proximal was blocked by vascular clamp temporary,And then,we give a little cut at the top of the ligatured femoral artery to make a small incision,a balloon catheter was inserted into the iliac artery of rabbits through the right femoral artery,and the aorta was injured by engorged balloons.Anesthesia was induced with SuMaAn(0.1-0.15ml/kg).After the procedure the animals received intramuscular antibiotics for 3 days(80 IU/day-penicillin).Rosiglitazone was administered by oral gavage(0.5mg/Kg body weight/day) to therapy group..3.Blood chemistryBlood samples were obtained on first day of the experiment,one week and four weeks after balloon catheter injury.Clinical laboratory assessment included fasting serum glucose(FPG),total cholesterol(TC),triglycerides(TGC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(1DL-C) and high C-reactive protein(hs-CRP).Measurements were done using an automated system(Unicel Dxc 800;BECKMAN COULTER,American).The adiponectin level was measured with ELISA method,specific procedures was according with the instructions. 4.Quantitative histopathologyOne week and four weeks after the experiment,five rabbits of each group were sacrificed by air embolism and their iliac were sectioned and stained by HE staining histological analysis and immunohistochemical Histological analysis was performed by an experienced pathologist unaware of the RGZ treatment.The analyses was done with microscope.Histomorphometric parameters were performed by calculation of the intimal area(IA),medial area(MA),and intimal Proliferation index(IHI).The proliferation rate of smooth muscle cells PCNA was examined with immunohistochemistrical analysis,Tissue preparation and immunohistological techniques were performed according to the manufacturer's instructions.5.Apoptosis detectionSmooth muscle cells apoptosis was quantitatively analyzed by TUNEL assay as manufacturer's instructions.The analysis was done with microscope.(×400).The ratio of the apoptosis will be calculate and describe by figure and table.6.Statistical analysisAll values in the text,tables and figures are presented as as mean±SD.Data were compared using Anova one-way.The normality of the samples was tested by using Shapiro-Wilk tests.The Kruskal-Wallis and Mann-Whitney was used for non-normal samples.The Pearson correlation coefficient was used for qualitative or categorical variables.Statistical significance was indicated by a value of p＜0.05. Analyses were performed using SPSS version 13.0.Results1.Effect of rosiglitazone on expression of adiponectin and lipidHypercholesterolemia was successfully induced.Baseline glucose,total cholesterol,HDL-cholesterol,LDL-cholesterol,triglycerides and hs-CRP levels were equal in all groups before initiation of the diet.On one,four weeks after feeding hypercholesterol diets and balloon endothelial denudation,level of serum TC,TG, LDL-C and hs-CRP level had significant differences in model and rosiglitazone groups contrast with control group(P＜0.01 ),but the level of FPG have no significant difference.After one and four weeks of rosiglitazone intervention,we can see a great difference between model and therapy group(P＜0.01 or P＜0.05 ).Baseline APN level was equal in all groups at the beginning of the experiment,and were decreased in one week after experiment in model and rosiglitazone groups,but is more lower in model groups(1.44±0.31 vs 1.62±0.63 ug/ml,P =0.191),and these change is more significantly four weeks later(1.17±0.33 vsl.85±0.16 ug/ml,P＜0.01 ).2.The relationship between adiponectin and intimal Proliferation index The experimental results show that.As the increasing of total cholesterol, triglycerides and LDL-cholesterol after the experiment,the adiponectin level is decreased.There is a significant negative correlation between adiponectin and intimal Proliferation index,and is more apparently in four weeks after the experiment (r=-0.733,P=0.002),adiponectin also have a negative correlation with hs-CRP level(r=-0.860,P =0.000 ),It remind us that adiponectin involved in the pathophysiological process such as lipid metabolism disorder and endometrial proliferation.3.HistomorphometryAt different time after the experiment,the intimal area(IA),medial area(MA), and intimal proliferation index(IHI) of operation group were significantly increased compared with those of control group(P＜0.01) with HE staining and elastic fiber staining.Thickened intima,luminal stenosis,and lots of macrophages can be seen in model group,After one week or four weeks of rosiglitazone intervention,The intimal area,medial area,intimal Proliferation index of rosiglitazone group were significantly lower than those in model group(P＜0.01 or P＜0.05 ),nd these changes were more significantly in 4 weeks after balloon endothelial denudation.4.Immunohistochemistry and ApoptosisThere was no statistically significant difference in the percentage of animals with apoptosis between three groups.Only a small number of positive PCNA cells can be seen in normal tissues,The expression of PCNA and apoptosis rate of surgery group was significantly increased compared with that of control group(P＜0.01),after four weeks of rosiglitazone intervention,the apoptosis rate of surgery group was significantly increased(21.84±3.22 vs 36.25±3.79,P=0.000),and the expression of PCNA is decreased compared with that of model group(58.76±5.44 vs 21.36±3.89,P =0.000).and these changes were more significantly in 4 weeks after balloon endothelial denudation.ConclusionRosiglitazone could efficiently inhibit intimal proliferation and promotes the apoptosis of vascular smooth muscle cells during the restenosis after balloon injury, the mechanism of which may be related to the increasing expression of adiponectin.