Effects And Mechanism Of Rosiglitazone On Restenosis Of Rat Abdominal Artery After Balloon Injury

PrefaceSince the technique of percutanous transluminal coronary angioplasty (PT-CA) has been applied in clinic, distinguished therapeutic effect had been a-chieved. It has become one of the important means of treating coronary heart disease. With the unceasing improvement of the operation technique and the accumulation of operators experience, the success rate is increasing rapidly. But the high incidence rate of the restenosis ( RS) is still the major factor of influencing the long - term effect, which restrict this therapeutic method to be spread. Plentiful foundational and clinical studies indicate that the process of vascular RS after balloon injury is the reparative reaction of organism to injury . Multiple factors participate in the formation of RS. The precise mechanism of RS is still unclear completely now. Although a large amount of researches have been done, and the incidence rate of RS has been obviously decreased by the application of antilplatelet drug and implantation of drug eluting stent, there is still no kind of safe, effective, easily - taken, or effecting on multiple pathways drug to prevent and treat RS.RS after the ballon injury is the result of proliferation of vascular smooth muscle cells, accumulation of extracellular matrix ( ECM ) and vascular remodeling caused by multi - factors. ECM in the hyperplastic intima is synthesized and excreted by the VSMC after its immigration to the intima, while the immigration of VSMC is associated with matrix metalloprotease. During the process of formation of restenosis after vascular injury, multiple kinds of active substances and growth factors are released, and activated the transcriptional factors by specific receptors through intracellular signal transduction pathway, priming the express-ing of specific intranuclear genes and make the biological effects. Previous researches illuminated that extracellular signal regulated kinase ( ERK) signal transduction pathway is the important pathway of regulating the cellular proliferation by major growth factors and cytokines;the activation of ERK is one of the key factors of vascular restenosis after balloon injury. TGFpi/smad3 signal transduction pathway has multiple biological effects of participation in the cellular growth and differentiation, embryonic development of tissue and organs, repairing of injury, especially has the important effects on the expression of organic collagen and the aggregation and degradation of extracellular matrix. So intervention of TGF(3l/smad3 and ERKl/2 signal transduction pathway maybe the therapeutic targets of preventing and treating RS.Rosiglitazone (RSG) belongs to insulin sensitiveness - thiazolidinediones (TZDs). For the past few years, research on the mechanism of TZDs indicated TZDs were the high affinity ligand to Peroxisome proliferators activated receptor ( PPAR) y. PPAR-y can not only improve insulin resistance syndrome, but also inhibit the expression of vascular cell adhesion molecules on incubated endothe-lial cells, improved the adhesion function of endothelial cells;PPAR-y agonist can inhibit vascular smooth muscle cell (VSMC ) proliferation;PPAR-y agonists can inhibit VSMC migration through down regulation of the expression of matrix metalloprotease - 9 (MMP - 9);activation of PPAR-y can inhibit inflammation, stabilize artherosclerotic plaque. These changes above mentioned are associated with the vascular restenosis after angioplasty. It revealed that RSG possibly become the drugs to treat and prevent RS by multiple pathways. At present, there are no reports about the in vivo researches on the effects of RSG on TGF(31/ smad3 signal transduction pathway and expression of ERKl/2 during RS process after angioplasty. In this study, we used the model of vascular restenosis established by balloon injury of rat abdominal aorta and observed the effects of RSG on RS in vivo, detected the changes of MMP9, TGFpi/smad3 signal transduction and extracellular signal - regulated kinase ( ERKl/2) expression in artery after balloon injury. The aim was to invest the mechanism of RSG to prevent and treat RS after injury, to search the new therapeutic targets of RS.Material and Methods1. Animal model(1) Experimental animals and groupsHealthy male Wistar rats , weight 300 - 350g, providedly by CMU trial animal department, divided into 3 groups randomly. Treatment group ( n = 30) : rats were injured by balloon and treated with RSG 5mg kg"1 d"1 intragastric administration;Balloon injury group ( n = 30) : rats were injured by balloon;sham -operated group(n =30). Each group was subdivided into ld,3d,7d,14d and 28d subgroups according to different interventional time point. There were 6 rats in each subgroup.(2) Construction of restenotic animal modelsA 2.5 x 20 mm PTC A catheter was inserted from left common carotid to abdominal artery to denude the arterial wall at a pressure of 6kPa by pulling and rotating the fluted balloon from distally to proximally over a 14 cm long segment foe three successive times.(3) Sample collectionAnimals were executed by intraperitoneal injection of 10% Chloral Hydrate on day 1,3 ,7 ,14 and 28. The abdominal artery was selected rapidly, perfused by physiological saline, divided into segments and putted in 4% paraformalde-hyde fixation liquid and nitrogen jar freezing and then conserved in -70X1 deep freezing refrigerator.2. Experimental method and detection index(1) Morphology of aortaThe pathologic changes of vascular wall at different time point after injury and the effect of RSG was observed by light microscopy. Thickness and area of neointima and media , lumen area and cross - sectional area bounded by external elastic lumina(EEL) and internal elastic lumina( IEL) at different time point after balloon injury were measured by computer images analysis technology.(2) Imrnunohistochemistry detectionImmunohistochemistry technique was used to detect the expression of colla-gen, MMP - 9 and PCNA protein in arterial wall after balloon injury and observe the effects of RSG on them.(3 ) RT - PCR: reverse transcriptase - PCR was used to measure the changes of PPAR7,MMP -9 and TGF - pi mRNA expression of rat abdominal aorta after balloon injury.(4) Western blotting: western - blot was used to measure the protein expression of TGFpJl, phosphorylated smad3 and phosphorylated ERK1/2 in injured vascular tissue.3. Statistical analysisAll data were expressed as the means ± SD, the one way AN OVA and Dun-nett test was carried out using SPSS statistical software.ResultsPARTI The expression of MMP - 9 and collagen in abdominal aorta after balloon injury and the effects of RSG on them1. The neointima was formed at 3 day after balloon injury , become thick at. 7th dayafter injury and continuously to be thick during 28days. The thickness of the media increased at 3 rd day ,was maximal at 14th day. AT 28th day the thickness of media was smaller than that of at 7' day. The neointimal area was increased at 7th day after injury, and increased gradually during 28 days, the lumen area was obviously declined at 14th and 28th day. The thickness and area of neointima was smaller in RSG group than that of injury group (P <0. 01). At 28th day,the neointimal area decreased 23. 5%in RSG group compared with injury group, the area of lumen loss was decreased 18. 8% in RSG group compared with injury group.2. The expression of collagen in the neointima was started from the 7 day after injury, was maximal at 28th day. The expression of collagen in the media was started from the 3 day, peaked at 7th day. The expression of collagen in neointima and media was reduced by RSG.3. The expression of MMP - 9 was up - regulated after balloon injury, peaked at 3 day. At 14 to 28* day, the expression decreased obviously tobaseline in each layer. The expression of MMP - 9 in the neointima was peaked at 7 th day The expression of MMP - 9 in neointima and media after injury was down - regulated by RSG.4. The expression of MMP -9 in aorta after balloon injury and PCNA was almost paralleled o There was positive correlation relationship between PCNA expression and MMP -9 expression in neointima and media (0. 928, 0. 866 re-spectivelly, P<0.01).PART2 The expression of TGF(Jl/smad3 signal transduction pathway in abdominal aorta restenosis after balloon injury and the effects of RSG on them1. The expression of TGFpimRNA was up - regulated after balloon injury at 3 day compared with sham - group, the expression level was gradually increased ,peaked at 14th day. The expression tendency of TGFpimRNA in RSG group was the same as the injury group ,but the expression of TGFpimRNA was down - regulated by RSG.2. The expression of TGFpi protein was very low in sham - operated group . The expression of TGFpi protein was increased predominantly in injury group at T day after balloon injury compared with sham - operated group, peaked at 14th day , decreased at 28th day significantly. The expression of TGFpi protein was down - regulated by RSG.3. The expression of smad3 protein was very low in sham - operated group . The expression of smad3 protein was increased predominantly in injury group at 7' day after balloon injury compared with sham -operated group, peaked at 14' day , decreased at 28' day slightly. The expression of Smad3 protein was down - regulated by RSG.4. There almost was no expression of PPAR7 in normal vessels The expression of PPAR-y protein was increased in injury group at 3 day after balloon injury , peaked at 7' day , decreased gradually from 14' day to 28' day . The expression of PPAR7 protein was down - regulated by RSG.5. There was negative correlation between TGFpi and lurninal area(r = -0.719, P<0.01). The expression phase of TGFpimRNA and intima collagen expression was almost the same.PART3 The expression of ERKl/2 signal transaction pathway in abdominal aorta restenosis after balloon injury and the effects of RSG on them1. The expression of P - ERKl/2 protein was increased in injury group at 3 day after balloon injury , peaked at 7' day , decreased at 14' day and 28' day slightly . The expression of P - ERKl/2 protein in vessels after injury was down -regulated by RSG, at 7' day , the expression of P -ERKl/2 protein in RSG group decreased 15. 9% and 1.1% respevtively compared with injury group, P<0.01.2. The expression of p - stat3 protein was up - regulated slightly after balloon injury at 3 rd day compared with sham - group, peaked at 7 th day, decreased gradually from 14l day to 28' day. The expression of stat3 protein was inhibited by RSG, it was predominant at lx day by decreasing 1.1% , P<0.01.3. The expression of MMP -9mRNA was up - regulated after balloon injury, peaked at 7' day. At 14' to 28' day ,the expression decreased gradually to baseline in each layer. The expression of MMP - 9 after injury was down - regulated by RSG, P<0.05.4. The expression phase of P - ERK, p - stat3 protein and MMP - 9mRNA were almost the same;The coefficient correlation between P - ERK and intima PCNA was 0.929, P <0.01;There was positive correlation relationship between P - ERKl/2 and p - stat3 protein expression(r =0. 873,0. 875 respectively, PConclusions1. MMP - 9 participates in the formation of RS which mainly are associated with VSMC immigration, proliferation to intima.2. RSG can relieve intimal hyperplasia and RS after balloon injury. Its mechanism maybe associated with down - regulating the expression of MMP - 9, inhibiting VSMC immigration, proliferation and extracellular matrix accumulation.3. TGFpi/smad3 signal transduction pathway in vessels is activated afterballoon injury. RSG inhibits the activation of TGFpi/smacB signal transduction pathway during RS process .4. RSG inhibits the activation of ERK1/2 signal transduction pathway during RS process.5. RSG prevents and treats RS after balloon injury through multiple mechanisms. Intervention of TGFpl/smacB and ERK1/2 signal transduction pathway maybe the therapeutic targets of preventing and treating RS.