The Effects And Mechanism Of DIM On Restenosis After Carotid Artery Rabbit Balloon Injury

Coronary heart disease(CHD)is a common disease,which severely threatens human health and affects life quality of patients .Main various measures used for the treatment of CHD includes pharmacological agents,percutaneous transluminal coronary angioplasty(PTCA) and coronary artery bypass grafting(CABG). Angioplasty is an effective, minor-wounded method for CHD. The incidence of restenosis(RS) which confirmed with coronary angiongraphy over the first six months following angioplasty remains 30%-50%.But restenosis is still the most important long-term limitation.Therefore how to reduce the ratio of restenosis is challenges both in basic and clinic research. Pathological studies have suggested that restenosis is correlation to vessel chronic elastic recoil, thrombus formation in situs, vascular smooth muscle cells(SMCS) migration and proliferation, and excessive extracellular matrix (ECM)synthesis. Cells in the restenostic plaques were mainly composed of VSMCs. Neointimal hyperplasia was formed by VSMCs migration from vessel media to subendothelium and proliferation. So, it is a key step to inhibit VSMCs proliferation for preventing restenosis.The process of cells proliferation is bound to follow by cells apoptosis in cytology. Growth factors-related and bcl-2 play an important role in the VSMC proliferation. If apoptosis of VSMCs is induced in the duration of restenosis,certainly neointimal formation will be decreased.In the end restenosisis may be prevented.The search for an appropriate agent which inhibits VSMC proliferation and promote VSMC proliferation will contribute to better treatment of restenosis.DIM is one of the active components of I3C, and it has various pharmacological activities that may affect many tumor cells. In addition, it's reported recently that DIM can inhibit proliferation of tumor cells.It is a much better agent used for induction of cancers,via suppressing some cell growth factors and cancer cells expression to inhibit cancers proliferation and induce cancer cells apoptosis. And cell apoptosis is found in human atherosclerosis plaque and in the process of restenosis. However, little is known about its antiproliferative mechanisms, and there is no directive evidence about its effect of preventing restenosis.We wondered whether DIM may be inhibit restenosis after angioplasty.So we performed the initial experiment of DIM on prevention of restenosis.Objective①To create the common carotid artery injury model of rabbit and to evaluate the effccts of DIM on neointimal proliferation and vascular remodeling after percutaneous transluminal angioplasty( PTA),and confirm DIM may prevent and treat rabbit' s experimental restenosis after PTA.②To investigate the role for some growth factors-related in restenosis experiment. Here ,we confirm the expression and significance of GF-related during restenosis.③To demonstrate DIM may induce VSMC apoptosis to prevent RS after artery injury,via downregulation of gene bcl-2 expression. We discuss the effect and significance of cell apoptosis gene during restenosis. .④To assess the safety of DIM on neointimal proliferation of rabbit artery after balloon angioplasty.Materials and MethodsRestenosis models of carotid artery after balloon injury was established in rabbits.30 rabbits were randomly divided into 4 groups including sham-operated group,model group,low-dose DIM group and high-dose DIM group. DIM was given to the rabbits in low-dose treatment group[2mg/㎏﹒d ] and high-dose treatment group[8mg/㎏﹒d ]once a day from 3 days before operation to 4 weeks after operation,the two treatment groups were administered with intraperitoneal injection of emulsified DIM,while the other two groups with saline at the same volume. all rabbits were killed after 28 days and carotid arteries were removed. With HE staining,automatic image analysis and immunohistochemistry,We observed artery morphology and measured area and thickness of arterial intima and media,examined platelet derived growth factor﹙ PDGF﹚,transfer growth factorβ1﹙TGF-β1﹚,vascular endothelial growth factor﹙VEGF﹚ and basic fibroblast growth factor﹙bFGF﹚.the expression of bcl-2 gene was observed by in situ hybridization(ISH)technology and computer-assisted picture analysis system .To evaluate the safety of DIM in experiment by assaying serum alanine aminotransferase and aspartic aminotransferase by examinating serum in all rabbits.ResultsAfter one week of diet,30 rabbits were balloon injured on carotid artery, then they were randomly separated into sham-operated groups,low-dose groups( 2mg·kg一·d一)and high-dose groups (8mg·kg一·d一)and model groups. At the end of 4 weeks after carotid injury,the rabbits were killed in groups. The arteries were examined with histomorphometrical method, examined PDGF , VEGF , TGF-βand bFGF With immunohistochemical method and bcl-2 with ISH.1 The efects of captopril on neointima hyperplasia and remodeling during restenosis angiography.1.1 Pathological changes The status of the models'arteries after the operation is thrombi-generated in the vessel, as well intimal hyperplasia and lumen stenosis;hyperplastic , shed and partly internal-plastic-membrane-broken in tunica intima .The status is obviously hyperplastic in tunica intima ,antrum-narrow,intimal and medial elastic tissue broken,especially wounded model groups after PTA,all the arteries are narrow and there are SMCs in tunica all intima mainly; there was no obvious difference in area and thickness of intima between model groups and low-dose groups; slightly thickened in tunica adventitia Lumen area,EEL perimeter,IEL perimeter of model group were decreased significantly in high-dose groups than those of model groups.1.2 Changes of the intimal, medial and adventitial thickennessThe neointimai thickness increased 28 days after injury.There was no significantly change for both medial and adventtial thickness in model groups. There were also no significantly differences among the intimal,medial and Adventitial thickness between the low-dose group and model group; however,Compared with that of model groups ,the thickness,ECM and the size of lumen were markedly declinded in high-dose group (P <0.01).1.3 Changes of the intimal, medial and adventtial areaThe neointmtal area was increased the 28 days following injury in model groups.Compared with that of model group , the intimal area,media area and intima-to-media ratio increased obviously in high-dose group (P <0.01),but there was no statistical significant difference between model group and low-dose groups,high-dose group also showed less extent of restenosis of injured arteries in comparision with the low-dose groups and model group significantly,suggesting the model is successful.2 The expression and significance of growth factors during restenosisImmunohistochemical assay in transfected tissues showed a small quantity of positive cells with VEGF,PDGF,TGF-βand bFGF mRNA can be found in the advemitia and media in four groups. The number of positve markedly increased in the neointima at 28 days after balloon injury in model groups. There were no obvious differences for the expression of VEGF,TGF-β,PDGF and bFGF mRNA in the vessel wall between low-dose groups and model groups ,but light density of VEGF,PDGF,TGF-βand bFGF mRNA in intima in high-dose groups less than That of model groups (P <0.01). so DIM may decrease the positive index of VEGF,TGF-β,bFGF and PDGF (P<0.01).3 The effect of DIM on the expression of apoptosis geneThe results of in situ hybridization(ISH)showed that The increased production of bcl-2 was found in proliferation intima and media of all groups, intensive density of bcl-2 in model groups more than That of high-dose groups (P <0.01):there was not significantly difference between model groups and low-dose groups ( p>0.05). Conclusion1 DIM can effectively inhibits intima hyperplasia,which is mainly characterized with the proliferation and migration of smooth muscle cells .DIM effectively prevents intimal thickening and area. This result suggestes that DIM may play a positive role in the prevention of restenosis after PCI.2 The therapy of DIM can reduced the rabbit carotid artery neointimal hyperplasia induced by ballon injury by decreasing The expression level of growth factors,also DIM can improve constrictive vascular remodeling.3.bcl-2 may participate in regulating of VSMC apoptosis .So DIM can promotes SMC apoptosis and decreases The expression of Bel一2 in intima and media.4 The models of vascular restenosis established by balloon intimal injury of rabbit carotid arteries were successfully replicated, and proved to be rational and scientific.lntimal proliferation and vascular remodeling are important pathologic pathogenesis of vascular restenosis. The formation of vascular restenosis was determined by the unbalance of intimal proliferation and vascular remodeling.The both together resulted in lumen narrowing.5 DIM was little toxicity during performing experiment,so DIM was basically safe for animals.