| Although many significant advances have been made in diagnosis and treatment of coronary heart disease, it is still has extremely high mortality rate and morbidity in the world, especially in patients with acute coronary syndrome which is still the important killer to present a threat to human health. Atherosclerosis (atherosclerosis, AS), as the main pathology of coronary heart disease, has complex causes which are closely related to genetic risk factors, hyperlipidemia, smoking, diabetes, age, etc. The serious detriment of AS to human health provoked people’s tremendous efforts to explore the pathogenesis and progress of the disease. And the current awareness of the progress of AS mainly from various researches, including human pathological anatomy and cell biology based on animal models. There are many theories for the pathogenesis of AS, containing lipid infiltration theory, thrombosis doctrine and the doctrine of smooth muscle cloning, etc. In recent years, most scholars support the "endothelial damage response doctrine", considering that AS is a chronic reaction to endothelial cell damage and promote the progress of the disease through the interaction between the normal existence composition of the arterial wall and the oxidative modification lipoprotein, monocyte-derived macrophages, T lymphocytes etc. Core concepts of endothelial damage response doctrine include:①chronic injuring of endothelial cells, sometimes being associated with dysfunction, cause increased vascular permeability, monocyte adhesion and thrombus formation;②cholesterol-rich LDL (low density lipoprotein, LDL) accumulates in the blood vessel wall gradual;③oxidative modification of lipoproteins (eg LDL into ox-LDL) promote the formation and development of atherosclerotic plaques;④blood mononuclear cells move into the intima,then further transforme into macrophages and foam cell⑥activated cells (such as macrophages, smooth muscle cells, etc.) release of various factors;⑦intimal smooth muscle cell hyperplasia, proteoglycans and collagen accumulate;⑧lipid accumulate inside and outside of the cell. Histological studies found that acute coronary syndromes were often caused by the rupture or erosion of vulnerable plaque to the secondary thrombosis.Macrophage-derived foam cells, the thickness of fibrous cap and plaque hemorrhage play an important role in this process, which has been proven in clinical practice. In order to describe the nonobstructive atherosclerotic plaques with the tendency of rupturing, Muller put forward the concept of "vulnerable plaque"(vulnerable plaque, VP) in1989, pointing out that such plaques typically have large lipid core, thin fibrous cap and plaque macrophage infiltration. As the accumulation of research, the concept of vulnerable plaque was gradually recognized by scholars, Due to the thin fibrous cap and large lipid core, this type plaque is prone to rupture, bleeding under the influence of shear stress, physical and chemical factors, then lead to the formation of local thrombosis and acute occlusion in severe situation, and finally result in ischemic necrosis of tissue, which increase the occurs of clinical acute cardiovascular events. The adequate study of the progressive atherosclerotic lesions may provide important clues to our clinical treatment, such as what methods or drugs can be used to the therapy of stabilizing plaque. The models of atherosclerotic plaque include classic aortic balloon injury+high-fat diet, high-fat diet alone, immune damage+high-fat diet, knockout mouse models, etc, which provide a good platform for our in-depth understanding of atherosclerosis. But the above methods of modeling have a different degree of constraints, such as operation difficulty, plaque morphology differences and a high expenditure. We need to establish an experimental animal model simple and similar to the formation of human. Based on the doctrine of endothelial injury, we try to establish an animal model of rabbit carotid atherosclerotic vulnerable plaque by endothelial injury caused by low-temperature gas and a high-fat diet, which provides a better platform for our study.CD147, also known as a matrix metalloproteinase inducer, is a trans-membrane glycoprotein that is widely expressed and highly glycosylated. It is a member of the immunoglobulin super-family, originally found in lung cancer. CD147is a matrix metalloproteinase (matrix metalloproteinases,MMPs) upstream regulatory factors, which could induce the generation of MMPs.in atherosclerotic plaques, excessive CD147by macrophages, foam cells, endothelial cells can stimulate the expression of MMPs excessive production which results in extracellular matrix synthesis and degradation imbalances and weakened the strength of the plaque fibrous cap making plaques prone to rupture, have an important role in the occurrence and rupture of the vulnerable plaque.The CyPA as one secreted factor induced by oxidative stress, has potentially chemotactic for endothelial cells/monocyte/neutrophils. Peter Seizer et al, in the study of the differentiation progress of CD34+cells into foam cells, found that CD147/MT1-MMP/MMP-9was up-regulated and CyPA released into the supernatant to promote the secretion of MMP-9. Besides, CyPA inhibitors can reduce the expression of MMP-9in the process of the cellular differentiation. It indicated that there was a certain regulation for CyPA to the expression of MMP-9. Endothelial cells and macrophages could express CD147/CyPA, there may be interactions between CD147and CyPA induce both ERK and NF-KB pathway activation in macrophages which leads to an increase of MMP-9expression levels. MMPs, MMP-9in particular, which degrade collagen and elastin, could weake the plaque fibrous cap and increase AS plaque instability.Based on the model, the structure of the plaques were analyzed to explore the feasibility of gas thermostat damage+high-fat diet rabbit carotid atherosclerotic vulnerable plaque; Analysis of CD147, CyPA, MMP-2, MMP-9distribution expressed in plaque by immunohistochemical staining.Maenwhile ShRNA lentiviral vector was used to transfect monocytes/macrophages to closed CD147was to explore the effect of CD147on the chemotaxis and MMP-9expression of mononuclear/macrophage induced by CyPA.Objective 1.Animal models of rabbit carotid vulnerable atherosclerotic plaque were made using continuously constant low-temperature gas-induce endothelial injury, and combined with high-fat diet, then explored its feasibility.2. Analysis was made about the morphology of vulnerable atherosclerotic plaque, expression distribution characteristics and serological changes of CD147, CyPA, MMP-2, MMP-9.3. ShRNA lentiviral vector was used to transfect monocytes/macrophages, which for blocking CD147to observe the interactions of CD147and CyPA in the produce of MMP-9.Method1.Animal models establishment. There were24healthy pure-bred male New Zealand white rabbits which were divided into three groups according to simple random method. group A was the blank control group: normal diet, group B was the Sham-operated group:High-fat diet, group C was temperature-controlled gas injury group:cryogenic gas damage+high-fat diet. High-fat diet for7days, the group C was injured on the right carotid intima using the output device of low-temperature gas. B group was given simple operation, without damage of intima.After the operation, group B and group C were kept feeding on high-fat diet for12weeks, group A was always normal diet.2. Serological indicators: peripheral blood was collected in the rabbit ear artery respectively before and after13-week high-fat diet.(before the end of the experiment). Then, using enzyme-linked immunosorbent assay (ELISA), we detected the level of lipids, CRP, matrix metalloproteinase2, matrix metalloproteinase-9, CD147, CyPA and etc.3. Histological sections:All animals were sacrificed after14weeks. Then paraffin-embedded and frozen sections were kept for HE staining, Masson trichrome staining and elastic fiber staining. RAM11, CD147, CyPA, MMP-2, TIMP2monoclonal antibody were applied for animal immunohistochemistry staining.4. CD147shRNA lentivirus vectors highly suppressing the CD147expression on monocytes/macrophage were successfully designed and synthesized according to the sequence of CD147mRNA from rabbits.The monocytes/macrophages isolated from peripheral blood in rabbits were divided into blank control group, negative control group or CD147shRNA lentivirus group, respectively. The monocytes/macrophages of negative control group were transfected by blank lentivirus and CD147shRNA lentivirus group by CD147-shRNA Lentivirus. The transfection results were observed by fluorescence microscopy, membrane CD147detected by flow cytometry.The levels of CD147mRNA or protein were evaluated in each group.Transwell chambers were used to detect the chemotaxis of mononuclear/macrophage induced by CyPA. MMP-9expressions of mononuclear/macrophage in three groups treated with CyPA for24hours were detected.5. Statistical analysis:SPSS13.0software was applied for data’s statistical description and analysis. Measurement data was presented by mean±SD. One-way ANOVA was used for the comparison of multiple samples. LSD test was used for the multiple comparison between the multiple samples under the condition of homogeneity of variance. However, under the circumstances of heterogeneity of variance, Dunnett’sT3was used to compare multiple samples. Paired t test was employed for the overall comparison of two paired samples. P<0.05was considered statistically significant.Result1.Rabbits generally grow well. Except a New Zealand white rabbits in Group C died of the anesthesia overdose and one in Group B died of diarrhea, then the remaining animals completed the experiment, the data derived from22animals was used for analysis. There are8animals in Group A,7in Group B and7in Group C.2.The rabbit carotid atherosclerosis established in our study in line with the progress characteristics of vulnerable plaque, such as thin fibrous cap, less collagen fibers, primarily consisting of endothelial cells and thin layer of smooth muscle cells, the scattered lipids presented at the bottom of the intima, lipid pool partly formed by the scattered lipids. The space between the lipid pool and the intima was infiltrated by macrophages and foam cells with a variety of source. There were visible fissures in some of plaque fibrous cap and small scattered thrombosis. These resulted confirmed that the characteristics of vulnerable plaques in our model was similar to that of human.3. According to plaque analysis, we found that there were a large number of plaques in macrophages, which was the major sign of active inflammation. Macrophages can degrade extracellular matrix and weaken the fibrous cap by phagocytosis or release of plasminogen activator, which is one of the characteristics for plaque with a tendency to rupture. New-born microvessels were visible in the outer membrane, which provided a way for inflammatory cells into the plaque. Then, the fibrous cap got thinner, consisting of a single layer of endothelial cells and a small amount of smooth muscle cells. Masson trichrome staining displayed a lack of collagen fibers in the fibrous cap. The fibrous cap was the thinnest in the plaques corner and the junction of the normal vessel wall and the plaques. The fibrous cap was prone to rupture due to the pressure derived from the systolic and diastolic pressure and the coronary blood flow shear stress. No significantly large lipid core in the plaque, which is related to a short period of high-fat diet model, but scattered lipid particle can be visible, and serious myxoid change existed in the plaque, which induced expansion and instability of the plaque. In addition, part of the endothelial cell lost, demonstrated as surface erosion, scattered fibrin aggregation, red blood cells and platelets wrapped. Furthermore, from the results of HE staining, we can see some plaque fissure extending to the depth of plaques, the defects in thelocal fibrous cap and a small amount aggregation of platelet, red blood cells, and fibrin. Finally, we observed severe stenosis. So we considered that in such models, cold gas not only damage the endothelium, but aslo vascular smooth muscle, the deep tissue membrane.4. Immunohistochemical analysis.Blank control group:The vascular structure was integral. In the intima, there were no macrophages except some scattered macrophages, a small amount expression of CyPA and CD147with uniformly distributed and no significant expression of MMP-2/MMP-9. In tunica media vasorumta, there were no macrophages and expression of CyPA, CD147, MMP-2, MMP-9.Sham-operated group:the results of immunohistochemistry of artery with no plaque were similar to white and red control group. The formed plaque was small, with fatty streaks change and rich of macrophage. There were scattered expression of CyPA, CD147, MMP-2and MMP-9. In tunica media vasorumta, macrophages occasionally expressed outside the internal elastic panels, with smooth muscle disorder.Temperature-controlled gas injury group:plaque was rich in macrophages and macrophage-derived foam cells. The expression and distribution of CD147and CyPA were banded and had a total regional, MMP-9and MMP-2in the whole plaque were scattered expression, the more expression of plaque near the lumen, some elastic plate appeared rupture and dissolution. The smooth muscle cells in tunica media vasorumta integrated with plaque component. The expression of macrophages was rich. We also observed the expression of CD147, CyPA, MMP-9, and MMP-2with no obvious pattern.5.Serum indicatorThe results of serum MMP-9test showed that, before the experiment, serum MMP-9levels in three groups of rabbit had no significant difference.(P>0.05). After14weeks, serum MMP-9levels in the blank control group had no significant difference when compared with the same group before the experiments (P>0.05). And in sham-operated group and the temperature-controlled gas injury group, the serum MMP-9levels had significant difference compared with the same group before the experiments (P<0.05). The serum MMP-9levels in sham-operated group was significantly lower than the temperature-controlled gas injury group (P<0.05).The results of serum CRP test showed that: Before the experiment, the serum CRP levels in three groups of rabbit had no significant difference (P>0.05). After14weeks, the serum CRP levels in the blank control group had no significant difference compared with the same group before the experiments (P>0.05). And in sham-operated group and the temperature-controlled gas injury group, the serum CRP levels was significant higher than the same group before the experiments (P<0.05). The serum CRP levels in sham-operated group was significantly lower than the temperature-controlled gas injury group (P<0.05). The results of serum CD147test showed that: Before the experiment, the serum CD147levels in three groups of rabbit had no significant difference (P>0.05). After14weeks, the serum CD147levels in the blank control group had no significant difference compared with the same group before the experiments (P>0.05). The serum CD147levels in sham-operated group had a tendency to improvement but had no statistical difference. And in the temperature-controlled gas injury group, the serum CD147levels was significant higher than the same group before the experiments (P<0.05). The serum CD147levels in sham-operated group was significantly lower than the temperature-controlled gas injury group (P<0.05).The results of serum CyPA test showed that:Before the experiment, the serum CyPA levels in three groups of rabbit had no significant difference (P>0.05). After14weeks, the serum CyPA levels in the blank control group had no significant difference compared with the same group before the experiments (P>0.05). The serum CyPA levels in sham-operated group had a tendency to improvement but had no statistical difference. And in the temperature-controlled gas injury group, the serum CyPA levels was significant higher than the same group before the experiments (P<0.05). The serum CyPA levels in sham-operated group was significantly lower than the temperature-controlled gas injury group (P<0.05).Lipid changes:After14-week high-fat diet TC, TG, LDL and other indicators in group B and group C was significantly higher than former(P<0.05). The blank control group had no significant difference before and after the high-fat diet (P>0.05).6.The protein expression of MMP-9was no significant difference in the control group and lentiviral blank group (P<0.05). The protein expression of MMP-9in CD147lentiviral group had statistical difference significance when compared with the control group and the NC control group(P<0.05).Conclusions1. We successfully made a rabbit model of carotid vulnerable atherosclerotic plaque using a method of endothelial damage with thermostatic gas continuous stimulation combined with high-fat diet, morphological analysis results were in line with the progress of atherosclerotic lesions and similar to human vulnerable plaque.2. The distribution of CD147and CyPA in vulnerable plaque has a total regiona. CyPA may play a catalytic role in generating the MMPs. A large number of macrophages and foam cells wan found gathered in vulnerable plaques and the expression of MMP-9and MMP-2was significantly increased.3. After blocking CD147, the production of cyclophilin-induced MMP-9was reduced, indicating that CyPA and CD147had interaction in the process of MMP-9generation. |
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