| PrefaceAccording to epidemic research , CHD will become one of the key disease to the people lived in 21 centry,threatening people' s life ,hindering economic development. Despite the technique of PCI has become the main effective remedy of CHD, restenosis rate is still high (about 10% -60% in 6 ~9 months after coronary angioplasty) . Restenosis is a kind of repairment reaction of local blood vessels after injury . Migration and prolifration of VSMC and vascular remodeling mediated by abnormal expression of many cell factors and growth factors are the major manifestations of restenosis. Recently, it has been focused many attentions upon the progress on the relations between Rho - kinase and restenosis . Some scholars has attempted to explore methods of restenosis prevention and treatment through the pathway of Rho - kinase. Rho/Rho - kinase is one of the intracellular signal polypeptides functioned as molecular switch in signal trans-duction. Recent studies have shown that Rho - kinase has involved in the patho-genesis of restenosis by influencing the cellular signal transduction system through many pathways.This trial is to observe the expression of Rho - kinase and MCP -1 mRNA during vascular intimal proliferation and to investigate the effects of Rapamycin on the Rho - kinase and coronary intimal proliferation in swine model of coronary stenosis induced by IL - 1β.Materials and Methods1. Animal model: Forty male yokshire pigs weighing 25 ~ 30 kg , aging 2 ~3 months are randomly divided into 5 groups: ( 1) control group;( 2) model group I;( 3 ) model group II;( 4 ) model group III and (5 ) rapamycin group . After one week seeding, the animals were sedated with ketamine hydrochloride and valin and anesthetized with sodium pentobarbital. The animals were then in-tubated and ventilated with room air. Oxygen was supplemented via a positive -pressure respirator. Under aseptic conditions, a left thoracotomy was performed. The proximal segments of LAD and LCX were gently wrapped with cotton mesh that had absorbed 0.05ml of sepharose bead solution with or without recombinant human IL - 1(3. However, in the control group , the selective coronary artery was wrapped without IL - 1(3. The amount of IL - 1(3 is 3. 0|xg in model group I , 4. 5|xg in model group II , 6. 0|xg in model group IH and in rapamycin group. The pigs in control group and model group s had been feeded with common food after the operation. The pigs in rapamycin group had oral treatment with rapamycin 4mg/d from preoperation 3 days to postoperation 2 weeks.2. Coronary angiography : Two weeks after the thoracotomy , the animals were anesthetized and intubated. Selective coronary angiography were performed to study the stenosis conditions of coronary artery.3. Samples collection;After coronary arteriographic, the animals were killed with a lethal dose of sodium pentobarbital, the hearts were removed, the proximal segmen of LAD, LCX were reserved for histopathology and RT — PCR analyzing.4. Pathological analysis: Making wax sample with HE staining ,VG collagen staining , Verheff elastic fiber staining . The areas of vessel tube and inner -outer elastic plate were measured in photomicrographs with a computer — associated picture analysis system. The intimal and medial areas of the coronary artery were calculated.5. Expression of Rho - kinase, MCP - 1 mRNA: The mRNA level of Rho - kinase and MCP - 1 were detected by RT - PCR. The total RNA was isolatedfrom each Frozen tissue sample by the method of acid guanidinium thiocyanante /pheol/ehloroform extraction, then the cDNA of Rho - kinase, MCP - 1 were prepared using Bca - Best RNA PCR Kit. PCR was performed for amplification of specific cDNA. The PCR products were subjected to 2% sepharose gel elec-trophoresis, and measured by an imaging analysis system.Statistic AnalysisAll the data were expressed as mean values ± standard deviation. Statistical significance was determined by t test.Result1. Result of coronary angiography: The coronary artery were free from stenosis in control group, obvious stenosis in model groups and slight stenosis in ra-pamycin group. The stenosis rate and stenosis area of coronary artery in model group III are more obvious than in rapamycin group (65 ± 12% vs 28 ±9% ,24±5% vs58±9%,p<0.01).2. Histology result;Significant intimal thickening with newborn smooth muscel cells and many inflammatory cells infiltrated to the neointima and medial membrane were noted in model groups. The intimal thickening were markedly inhibited by oral rapamycin treatment.3. Result of RT - PCR: The mRNA level of Rho - kinase and MCP - 1 were significantly greater in model groups, and the increased expressions of Rho- kinase and MCP -1 were both prevented by treatment with rapamycin.Discussion1. About animal model:The study of vascular restenosis after coronary angioplasty is the ho.tspot in interventional cardiology presently. Ideal animal model is required either in researching the mechanisms or in exploiting new therapeutic methods. Majority of the previous experiments employed mouse, rat or rabbit to make the pathological changes of intimal proliferation of abdominal aorta, iliac artery, femoral artery or carotid by feeding fat diet or making damages by balloon expanding. These models have certain advantages (the price of the small animals is cheaper, the costof the experiment is lower, the process of making the model is more simple). But the disadvantages are also obvious. For example, because of the physiological characteristics of small animals are very different from human's, the experimental results came from small animals are difficult to applied to mankind. The anatomical structures of aorta , abdominal aorta, iliac artery, femoral artery and carotid are different from coronary arteries too. The reactions of these vessels to external impulses such as injury are also different from coronary arteries. In addition , because of the mechanisms of restenosis have not been completely illustrated , at present, the animal model which is completely similar to clinical practical situation in the matter of etiopathology can not be copied at the fundamental research , especially can not embody the inflammatory mechanism of restenosis. The making of animal model of vascular stenosis induced by inflammatory factors has not been reported in domestic. We make a small amount of IL - 1(3 absorbed to adventitia and bring about proliferation of intima and lumen stenosis in a short time . The success rate of model making is higher ( more than 80% ) , experimental period is shorter (1 ~ 2 weeks) and the extent of vascular stenosis is severe (average more than 60% ) . It similar in the pathological characteristics (the smooth muscle cells of the middle layer proliferate and migrate to the inner layer, the matrix element of inner layer increases , lipoid crystal deposits and inflammatory cells infiltrate) to the clinical real restenosis of coronary arteries. The successful establishment of the model confirmed that the inflammatory element plays a important role in the process of restenosis. The model is ideal in researching coronary artery lesions especially in researching the changes of inflammatory factors in coronary artery restenosis. The anatomical structures and physiological characteristics of miniature pig are similar to human's. The conclusions made from the experiments using miniature pigs as models are more practical. There are some disadvantages about this model. For example, the making cost is higher, the techniques used in model making are somewhat difficult to learn, and the antibodies are difficult to acquire when making studies correlative to immunology.2. The relationship of Rho - kinase,MCP - lmRNA expressions and intimal proliferation of coronay artery :Results of the experiment indicated that the expressions of Rho - kinase and MCP - 1 were both enhanced obviously during the process of intimal proliferation , confirmed that Rho - kinase and MCP - 1 are involved in the genesis of vascular stenosis. Rho - kinase is the serine/threonine protein kinase. There are a lot of evidences that indicates Rho - kinase not only interplays with many other vasoactive factors ( such as AT II , seronine, thrombin, ET - 1, norepineph-rine, extracellular nucleotide and urotensin II , etc ) , but also involves directly in the organization of actin, the regulation of cell adhesion and migration, contraction of smooth muscle cells, gene expression and cytokinesis. The genesis and development of the main cardiovascular diseases (including atherosclerosis, hypertension, coronary artery spasm, myocardial ischemia,etc) are all closely related with changes of those cell functions. According to literature report, the expression of Rho - kinase is increased at either mRNA level or protein level in rest-enosis. Rho - kinase take part in the genesis of restenosis through many different ways. For instance,Rho -kinase played a role in the contraction of endothelio-cyte, increasing the permeability of endotheliocyte , debasing the functions of endothliocyte as a barrier, therefore promoting the proliferation of vascular inti-ma. The main mechanism that Rho - kinase promoting the proliferation of intima is that the Rho — kinase inhibit the expression of p27. p27 is a nonspecific cyc-lin inhibitor , mainly acting on the cyclin - cdk complex of Gl period , making the cells stagnant at Gl period not splitting into S period. Rho - kinase may also increase the activities and gene expressions of MCP - 1 and TGF - pi, therefore promoting the forming of vascular restenosis. MCP -1 regulates early inflammatory reactions and subsequent hyperplasia of vascular middle menbrane, and TGF - j3l may regulates the fibrosis of the peripheral tissue of vessels and myocardial cells.MCP - 1 is a new kind of cytokine, secreted mainly by monocyte, macroph-agocyte, fibroblast, endotheliocyte and smooth muscle cell in certain condition , mainly acting on monocyte. Monocytes become macrophagocytes after they come into the tissues. Macrophagocyte secretes chemokines, growth regulatory factors, metalloproteinases and other hydrolase, they all took part in the genesis and development of arteriosclerosis and the regulation of intimal wall repairment aftervessel damages. All the genesis of arteriosclerosis and forming of restenosis are chronic inflammatory processs. The injured endotheliocytes could secrete manifold inflammatory factors. In them, MCP - 1 might attract monocytes in the blood migrate across endothelium , and differentiate into macrophagocytes. Macroph-agocytes then become foam cells in the effct of small and dense LDL - C peroxi-dation. Foreign scholars found , that MCP - 1 played a important role in the aggregation and activation of monocytes, can make monocyte adhere to endothelium and penetrate into its interspaces. Rho - kinase influence the contraction of monocytes directly,and then affect on the penetrating ability of monocyte.3. Inhibition of Rapamycin to expression of Rho - kinase, MCP - 1 mRNA and the proliferation of vascular intimal wall:Rapamycin is a white, solid crystal complex of macrolide, its molecular weight is 914 dalton. Rapamycin is lipophilic and very unstable in aqueous solution. Rapamycin metabolizes mainly through cytochrome p450 of liver micro-some.There is not a literature reporting whether Rapamycin influence expression of Rho - kinase in the course of intimal proliferation. This experiment observed that taking a given amount of Rapamycin orally can not only inhibit proliferation of intima mediated by inflammatory factors , reduce infiltration of inflammatory cells in the intima , but also reduce expression of Rho - kinase and MCP — lmRNA.Rapamycin is a powerful cell cycle regulatory factor arresting cell division from Gl phase to S phase through inhibiting the activity of cdk. Rapamycin firstly forms complex with FK506 binding protein (FKBP) after entered the cell. The target body of Rapamycin complex on the mammals cell is mTOR ( mammalian target of rapamycin) , This is a kind of macromolecule protein , the molecular weight is greater than 200000 , which is a kind of enzyme in signal transduction channel related to cell proliferation . After the function of mTOR was inhibited , the cell in Gl phase can not synthesize cyclin D2 , cyclinD3 , cdk4 , cdk6 etc required for continuing proliferation ,so can not turn into S phase .Conclusion1. IL - 13 mediated animal model of coronary intimal proliferation is a ideal model for the study of coronary intimal proliferative diseases.2. The expression of Rho - kinase mRNA is increased in coronary artery stenosis, suggesting that it may be a important mechanism in stenosis.3. The expression of MCP — 1 mRNA is increased in coronary artery stenosis, suggesting that it played a important role in stenosis.4. Rapamycin can inhibit the expression of Rho - kinase and MCP - 1 mRNA. |
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