| With the increasing development of living standards and the life stress, also the continuously changing life styles since 21th century, cardiovascular disease has become one of the major diseases threatening the health of people. The reports of WHO in 2011 showed that about 17.3million people died of cardiovascular disease each year around the world, and the mortality rate still remains increasing along the time. Atherosclerotic coronary heart disease (CHD), the most common cardiovascular disease, has been recently the main risk for the man’s life.Atherosclerosis, the most fundamental pathology of coronary heart disease, is due to a number of risk factors for long-term participation, trigger, and promote the progress of chronic inflammatory process of the arterial wall, from lipid deposition, intimal thickening, fatty streaks appear to stenosis, atherosclerotic plaques appear, need more than ten years to decades. It is still not clear about the atherosclerosis formation mechanism, so there are a great many hypotheses, including the theory of lipid infiltration, the thrombosis theory, the doctrine of vascular endothelial injury and inflammation hypotheses. What is now accepted by most people is the theory of vascular endothelial injury and inflammation hypotheses that, coupled with metabolism disorder of blood lipid and lipid sedimentary between the vessel wall and smooth muscle cells, endothelial is damaged and changed functionally by the stimulation of a variety of factors, causing the proliferation of smooth muscle cells; meanwhile the smooth muscle cells and mononuclear phagocytic lipid become foam cells, releasing thromboxane and other active substances after activated by the exposed collagen; These unusual ingredients with strong proinflammatory effect, resulted in a variety of inflammatory cytokines, leading to chronic inflammation in the plaque, the plaque area increased. thus the atherosclerosis is formed with the interaction of the various factors mentioned above. Atherosclerosis is a progressive and irreversible change. The existing studies show that the blood vessels, in childhood, will appear the early atherosclerotic changes like fatty streaks; and the symptoms will not be found until the middle age. Therefore, it is a hot topic how the atherosclerotic changes can be detected as early as possible so that the early prevention and treatment can be provided. The existing studies also show that endothelial dysfunction and vascular remodeling is the main pathological features of early atherosclerotic changes. Besides, the vascular remodeling can, to some extent, implies the endothelial dysfunction. Thus, it is of great significance for the early prevention and treatment to study the changes of vascular remodeling.Vascular remodeling is a chronic vascular changes triggered by the interaction of hemodynamic and humoral factor; it is a dynamic process, where the blood vessels are changing structures and functions to fit in the inner and outside environment. Vascular remodeling is not only a pathology of atherosclerosis process, but the structural basis of the continuous atherosclerosis. Its main pathological changes include the proliferation and migration of vascular smooth muscle cells and the degradation and re-distribution of the extracellular matrix.According to the structure and substrate specificity, Matrix metalloproteinases (MMPS), an protease superfamily activity-dependent of zinc ions, can be divided into collagenase, gum dissolved elements,membrane-bound MMPS and collagenase(MMP-2 and MMP-9). Collagenase can degrade extracellular matrix (ECM), which plays an important role in atherosclerosis and vascular remodeling process. ECM, an important part of vessel wall, is in the dynamic equilibrium between the constant synthesis and decomposition. Matrix metalloproteinases can constantly degrade ECM to make new tynthesis, and the degraded ECM leads to the excessive collagen deposition and promote vascular smooth muscle cell migration to the intima, resulting in intimal hyperplasia and then cardiovascular remodeling.Known as matrix metalloproteinase inducer, CD 147, one of the mambers of the immunoglobulin super family is a transmembrane glycoprotein widely expressed in a variety of physiological and pathological cells, including endothelial cells, hematopoietic cells and tumor cells. Cyclophilin A (cyPA), a cyclophilin protein, is an intracellular target of immunosuppressant cyclosporine. In addition to the regulation of immune function,it can also assist the cell signaling and regulate the cell survival and apoptosis. CD 147 is CyPA receptors and a matrix metalloproteinase inducer. With the interaction of macrophages and endothelial cells, the complexes of CyPA and CD 147 will promote matrix mmetalloproteinase release, degrade the extracellular matrix and cause the cardiovascular remodeling through activating the ERKl/2 and JAK/STAT signaling pathway.Meanwhile, the combination of cyPA and CD 147 can activate smooth muscle cells,cause its proliferation and then vascular remodeling.At present, the researches on cyPA-CD147 focus much on the field of cancer and rheumatism and little on the field of atherosclerosis. But no research can be found about the expression of cyPA-CD147 in the eraly pathological changes of AS and the vascular remodeling. This study aims to establish a rabbit model of atherosclerosis to observe the expression of cyPA-CD147 and vascular remodeling for the further analysis of the relation between them and thus the atherosclerotic changes can be early found and treated to reduce the morbidity, disability and mortality of cardiovascular disease.Objectives1. Atherosclerosis rabbit models were developed by injuring carotid intima with liquid nitrogen combined with high-fat diet to explore the condition of vascular remodeling.2. Analyzed the morphology of intima and plaque by HE staining, special staining for elastic fibert and masson staining. examed the expressions of CyPA, CD 147, MMP-9 in serum. and explore their expressions in the vessel by immunohistochemical staining. Detected the expressions of CD147mRNA and CyPAmRNA in the vessel.3. Observed the vascular remodeling indexes by using computerized image analyzing system. and explore the relationship between the expressions of CyPA, CD 147 and vascular remodeling by Correlation and regression analysis.Methods1.Establishing the animal model:32 healthy male New Zealand rabbits were randomly divided into control group, model group, cold-induced injury group, hyperlipid group (8 in each group). The control group with standard diet, and the hyperlipid group with hyperlipid diet, both of them without any injuried. Meanwhile, the model group and the cold-induced injury group were developed by injuring carotid intima with liquid nitrogen, then the model group combined with high-fat diet, but the cold-induced injury group with standard diet.2. Serologica test:The blood samples were collected before the experiment and the end of experiment from the rabbit ear artery. examed the expressions of CD 147, CyPA and matrix metalloproteinase-9 by using enzyme-linked immunosorbent assay (ELISA).3. Pathological observation:All rabbits were sacrificed after 13 weeks. then removed the corresponding artery we operating(nearly 4cm), Embedded in paraffin and sections, then stained with HE, elastic tissue staining and masson staining. Obersved the pathological changes by light microscope. Additionally, macrophagocyte/CyPA/CD147 monoclonal antibodies were applied for immunohistochemistical staining. The CD147 mRNA were detected by Reverse transcription-polymerase chain reaction (RT-PCR).4. Computer pathological image analysis system (Image-proplus 6.0) analyzed the related vascular remodeling indexes:analyzed external elastic laminal area (EELa), Internal elastic laminal area (IELA), Lumen area (LA), Minimum lumen diameter (MLD), Maximum intima thickness (MIT), Intima area (IA), Media area (MA), and Lumen stenosis (LS) by elastic fiber staining sections.5. Statistical analysis:The data obtained using SPSS 13.0 software for statistical analysis. Measurement data were presented by mean ±standard deviation. Multiple samples’comparision used One-way ANOVA test. If the Multiple samples’ comparision were under the condition of homogeneity of variance, LSD test was used to compare the samples between the multiple samples. Meanwhile, If the samples were heterogeneity of variance, Dunnett’sT3 test had been used. Paired t test was used between samples before and after the same group. P<0.05 was considered statistically significant.Results1. Rabbits generally grew well, except a rabbit in model group died due to the anesthesia overdose and one in cold-induced injury group died of bleeding, then the rest of rabbits were all completed the experiment,30 rabbits’data were got to analysis.2. Serologica test:The value of the CD147 in serum:There was no significant difference among the four groups before the experiment (P> 0.05). In the control group, compared with the beginning of the experiment, the value of CD 147 had no significant difference in the 13rd week (P> 0.05); in the model group, compared with the beginning of the experiment, the value of CD 147 was significantly higher in the 13rd week (P< 0.05); t in the cold-induced injury group, compared with the beginning of the experiment, the value of CD147 was significantly higher in the 13rd week (P< 0.05); in the hyperlipid group, compared with the beginning of the experiment, the value of CD147was significantly higher in the 13rd week (P< 0.05). In the 13rd week, There had significantly increased of the value of CD 147 in the model group comared with the other three groups (P< 0.05).The value of the CyPA in serum:There was no significant difference among the four groups before the experiment (P> 0.05). In the control group, compared with the beginning of the experiment, the value of CyPA had no significant difference in the 13rd week (P> 0.05); in the model group, compared with the beginning of the experiment, the value of CyPA was significantly higher in the 13rd week (P< 0.05); t in the cold-induced injury group, compared with the beginning of the experiment, the value of CyPA was significantly higher in the 13rd week (P< 0.05); in the hyperlipid group, compared with the beginning of the experiment, the value of CyPA was significantly higher in the 13rd week (P< 0.05). In the 13rd week, There had significantly increased of the value of CyPA in the model group comared with the other three groups (P< 0.05).The value of the MMP-9 in serum:There was no significant difference among the four groups before the experiment (P> 0.05). In the control group, compared with the beginning of the experiment, the value of MMP-9 had no significant difference in the 13rd week (P> 0.05); in the model group, compared with the beginning of the experiment, the value of MMP-9 was significantly higher in the 13rd week (P< 0.05); t in the cold-induced injury group, compared with the beginning of the experiment, the value of MMP-9 was significantly higher in the 13rd week (P< 0.05); in the hyperlipid group, compared with the beginning of the experiment, the value of MMP-9 was significantly higher in the 13rd week (P< 0.05). In the 13rd week, There had significantly increased of the value of MMP-9 in the model group comared with the other three groups (P< 0.05).3. Immunohistochemical analysisIn the control group, a small amount expression of macrophages scattered distributed in intima, but had not seen the expressions of CyPA, CD147, MMP-9. The vascular smooth muscle cells was integral in the tunica media, and there were no significant expressions of CyPA, CD147, MMP-9 and macrophages. In the model group, There were high expressions of CyPA and CD 147 in atherosclerotic plaques and hyperplastic intima. They distributed patchily and had a total regional. Compared with the macrophage antibody staining in the same vessel, the expression regions of CyPA, CD 147, MMP-9 were the same as the macrophage accumulation. In the cold-induced injury group, the intima mild hyperplasia, and the tunica media was integral, some scattered expressions of CyPA and CD 147 distributed in the hyperplasia intima, they also had a total regional distribution. In the hyperlipid group, some scattered expressions of CyPA and CD 147 distributed in the hyperplasia intima as the cold-induced injury group, and there had no significant expression in the no-hyperplasia intima.4. RT-PCRThe expression of CD147 mRNA:The expressions of CD147 mRNA in model group, cold-induced injury group, hyperlipid group all had significantly higher than the control group. Furthermore, the expressions of CD 147 mRNA in the model group was much higher than the other three groups (P< 0.05).The expression of CyPA mRNA:The expressions of CyPA mRNA in model group, cold-induced injury group, hyperlipid group all had significantly higher than the control group. Furthermore, the expressions of CyPA mRNA in the model group was much higher than the other three groups (P< 0.05).5.vascular remodeling indexsThe intima area, media area, and lumen stenosis in the model group, cold-induced injury group, hyperlipid group was significantly increased compared with control group (p<0.05). However, the Lumen area decreased significantly (P<0.05). There had significantly increased of the intima area in the model group comared with the other three groups (F=22.54,P<0.05). There had significantly decreased of the Lumen area in the model group comared with the other three groups (F=8.61, P<0.05). There had significantly increased of the Lumen stenosis in the model group comared with the other three groups (F=458.00, P<0.05).ConclusionsThe intimal hyperplasia and lumen stenosis existed in the atherosclerosis rabbit models. There were high expressions of CyPA, CD 147, MMP-9 in the serum, atherosclerotic plaques and hyperplastic intima. The higher expressions of CyPA, CD 147 the more severe intimal hyperplasia and lumen stenosis existed, so the vascular remodeling. |
PDF Full Text Request