Impact Of Rosuvatation On The Stability Of The Plaque And Expression Of CD147in Atherosclerotle Plaque Of Rabbit

At present, cardio-and cerebro-vascular diseases have become the most leadingthreat to human health for their high morbidity and mortality.Atherosclerosis (AS) is an important pathological basis of the occurrence and development of many cardio-and cerebro-vascular diseases (such as coronary heart disease, cerebral apoplexy).The atherosclerosis is caused by multiple factors (including hypertension, hyperlipidemia, hyperglycemia and smoking), which would induce the dysfunction of vascular epithelium, the proliferation and migration of smooth muscle cells, the inflammation of artery vessel wall, then the atherosclerotic plaque is formed, the inflammatory reaction exists throughout the occurrence and development of atherosclerosis.As the improvement in living conditions at present, the rates of many risk factors are increasing year by year, which cause seriously threat to human health and life and bring out the increase in the incidence and mortality of hyperlipidemia and atherosclerosis.At present, many scholars have done lots of research work in the pathogenesis of atherosclerosis, and put forward certain hypotheses, such as lipid theory, thrombosis, inflammation and damage theories.lt is critical to elucidate the pathogenesis of atherosclerosis for its prevention and treatment.Current research suggests that atherosclerotic disease is a chronic progressive inflammatory in artery wall, it is characterized by a lipid-rich atherosclerotic core composed of inflammatory cells aggregation and the fibrous cap covered on the surface. The pathology observation indicated that the plaque stability was closely related to the thickness of fibrous cap, inflammatory cell infiltration and the content of collagen tissues.But current studies suggested that plaque inflammation is the major factor leads to the thinning of the fibrous cap and the instability of atherosclerotic plaques. The inflammation can stimulate the activation of inflammatory cells in the plaque, which could overexpress and secret a large number of MMPs, resulting in the degradation of extracellular matrix including collagen, thinning of the fibrous cap and the plaque instability, then leads to the acute cardiovascular events.Matrix metalloproteinases (MMPs) is a general term of zinc dependent endopeptidases excreted from smooth muscle cells and macrophages.The activity of matrix metalloproteinases is largely determined by the thickness of atherosclerosis plaque fibrous cap and the collagen content. Therefore, the relationship between the matrix metalloproteinases and the stability of atherosclerotic plaque has become a research hotspot. matrix metalloproteinases in atherosclerotic plaque are mainly secreted by macrophages and smooth muscle cells, large number of Matrix metalloproteinases have been found in the macrophage accumulation zone of atherosclerotic plaque. The signal pathway of matrix metalloproteinases expression and functional regulation is not clear yet, besides the endogenous extracellular inhibitor, it is also regulated by the transcription and zymogen activation. The matrix metalloproteinase (MMPs) is the most important enzymes to degrade extracellular matrix (ECM), with the degradation of extracellular matrix can make the AS plaque changes from stable to unstable, which can lead to plaque rupture and cause acute coronary syndrome. Matrix metalloproteinases (MMPs), also known as matrixins are endoproteinases that degrading protein components of the extracellular matrix (ECM), cause its restoration and reconstruction. In this way, retain the appropriate structure of the ECM and basement membrane during both:physiological processes and pathological conditions. Changes in the structure of the ECM are accompanied by physiological processes such as embryogenesis, angiogenesis, apoptosis, and development and rebuilding of connective tissue. Under physiological conditions, the activity of MMPs is regulated at the transcriptional level, activation of proMMP precursor zymogens and interactions with endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs). The imbalance in the system of MMPs/TIMPs induces the development of many diseases, including cancer, fibrosis, arthritis, cardiovascular diseases, neurological and autoimmune disorders. The matrix metalloproteinases/tissue inhibitors of metalloproteinases system is involved in the regulation of extracellular matrix metabolism, which plays a crucial role with regards to maintenance of tissue integrity. During the occurrence of vascular pathologies including hypertension, the balance between proteases and their inhibitors is temporally destroyed. Even though there are conflicting data in the literature regarding the expression pattern of the vascular matrix metalloproteinase system, the occurring extracellular matrix turnover leads to the change of arterial mechanical properties. For example, hypertension plays crucial role in the formation of cardiovascular remodeling which seems to be characterized by an increase in extracellular matrix. Changes in arterial stiffness, a predictor for cardiovascular morbidity and mortality, are determined by alterations in vascular extracellular matrix due to hemodynamic, genetic, or other factors. It has become increasingly evident that blockade of the renin-angiotensin-aldosterone system and other pharmacological strategies, seem to be particularly effective in reducing vascular stiffness and collagen content in human and animal models. However, the relationship between extracellular matrix metabolism and the effects of therapy in hypertensive patients needs to be further explored in larger trials over a longer period of time.CD147is a highly glycosylated and widely expressed transmembraneglycoprotein, which is originally isolated from lung cancer cell LX-1, it is the member of immunoglobulin superfamily. CD147is a widely expressed plasma membrane protein that has been implicated in a variety of physiological and pathological activities. It is best known for its ability to function as extracellular matrix metalloproteinase inducer (hence the other name for this protein, EMMPRIN), but has also been shown to regulate lymphocyte responsiveness, monocarboxylate transporter expression and spermatogenesis. These functions reflect multiple interacting partners of CD147. Among these CD147-interacting proteins cyclophilins represent a particularly interesting class, both in terms of structural considerations and potential medical implications. CD147is expressed at varying levels in many cell types, including haematopoietic, epithelial and endothelial cells. It is up-regulated markedly on CD71-positive early erythroblasts, and in heart, placenta and thyroid tissues. Human CD147is a269amino acid-long protein that belongs to the type I integral membrane protein family with a predicted molecular mass of27kDa The N-terminal extracellular part of CD147consists of two immunoglobulin (Ig)-like domains that are heavily glycosylated. Endoglycosidase F treatment leads to a mobility shift from58kDa to28kDa approximately, suggesting that the majority of CD147glycosylation is N-linked. Recently, another form of CD147, containing an additional extracellular membrane-distal Ig-like domain, has been characterized. This form was shown to be responsible for the majority of homophilic CD147interactions.CD147has been shown to function as a signalling receptor for extracellular cyclophilins A and B and to mediate chemotactic activity of cyclophilins towards a variety of immune cells.The CD147molecule is a upstream regulatory factor of matrix metalloproteinases, it can induce the excessive production of MMPs by paracrine in cytoplasm, it also can promote the metastasis and inflammatory activities of immune diseases by degrading the collagen of extracellular matrix. It is a high glycosylation and widely expressed in hematopoietic and non-hematopoietic cell lines the transmembrane glycoprotein, belonging to a member of the immunoglobulin superfamily. Study show that CD147can induce the produce of the matrix metalloproteinase in smooth muscle cells, endothelial cells, monocytes and other cells,Current studies found that CD147could induce matrix metalloproteinase production in smooth muscle cells, endothelial cells, mononuclear cells and other cells, including MMP-1, MMP-2, MMP-3, MMP-9, Membrane Type1Matrix Metalloproteinase(MT-MMP), so we assume that CD147in the plaque may play an important role in the stability of plaque through the MMPs expression.Statins are commonly used for the treatment of cardio-and cerebro-vascular diseases at present, besides the lipid-lowering effect, its effects on the treatment of cardio-and cerebro-vascular diseases also benefite from the various non-lipid mechanisms, such as inhibiting the proliferation of smooth muscle cell, increasing the nitric oxide biosynthesis, improving the endothelial function, reversing the vascular remodeling, inhibiting thrombosis, anti-inflammatory effect, antioxidant and stabilizing the atherosclerotic plaques, these effects play an important role in the prevention and treatment of atherosclerosis. The word cholesterol derives from the Greek chole-(bile), stereos (solid), and-ol, the chemical suffix for alcohol. First identified in gallstones in1769, the modern name was begun by chemist Eugene Chevreul in1815. By1971Japanese biochemist Akira Endo began seeking compounds to lower cholesterol, and in1987Merck began marketing lovastatin isolated fromAspergillus terreus.The endogenous biosynthesis of cholesterol occurs through the mevalonate pathway HMGR is a highly regulated enzyme which catalyzes the rate-limiting step in cholesterol synthesis. Although HMGR is located within membranes of the endoplasmic reticulum, its catalytic domain remains active after it is cleaved from the transmembrane portion of the enzyme. The reduction of HMG to mevalonic acid involves the transfer of4electrons, using2molecules of nicotinamide adenine dinucleotide (NADPH) as a reductant in2steps. The carboxyl moiety of hydroxymethlyglutarate esterified to the thiol of CoA is first reduced to an aldehyde, and then to an alcohol. Statin drugs are highly efficient competitive inhibitors of HMGR.Rosuvastatin is a safe and efficacious lipid modifying drug in a broad variety of patient populations (men and women, irrespective of race) for treating multiple forms of dyslipidemia. Rosuvastatin reduces atherogenic lipoproteins and triglycerides and increases high-density lipoprotein cholesterol better than other statins. Compared to other statins, it has no excess signal for liver, skeletal muscle or renal toxicity. This is supported by evidence from both an extensive clinical trial program as well as post-marketing surveillance. The incidence of myalgia with this drug is comparable to that observed with other statins. Rosuvastatin does not depend on cytochrome P4503A4-dependent metabolism and has a favorable drug interaction profile. Care must be taken to reduce the dose of rosuvastatin in patients of Asian ancestry or with stage IV chronic kidney disease (severe renal insufficiency), as well as patients being treated with protease inhibitors or cyclosporine.The multiple effects of statins have attracted more and more concern, its anti-inflammatory effect has become the research hotspot now. Rosuvastatin is a novel type of lipid-lowering drugs commonly used in clinical practice, it can also play an important role in inhibiting the inflammatory reaction, improving endothelial function and stabilizing plaque besides the lipid lowering function.Currently, varieties of measures have been successfully used for the clinical prevention and treatment of atherosclerosis, such as antiplatelet and lipid-lowering therapies, but the limitations are still exist for the prevention and treatment of vulnerable or ruptured plaques, how to stabilize the vulnerable plaque is still the bottleneck and difficulty for prevention and treatment of cardio-and cerebro-vascular diseases. This research aims to establish the carotid atherosclerotic vulnerable plaque model in rabbit, to explore the relationship between the serum inflammatory factor levels, the expression levels of CD147in plaques with the stability of plaque, then elucidating the mechanism of Rosuvastatin in plaque stability.Object1. Establishing animal models of atherosclerotic plaque which are similar to Human plaque by cold-induced endothelial injury with liquid nitrogen combined with high fat diet fed,in order to lay a foundtion for basic and intervention research in vulnerable plaques.2. Based on the self-invented rabbit models of AS rupture plaque,we elucidate the relationship between expression of matrix matalloproteinase-2, matrix matalloproteinase-9,CD147and the stability of the atherosclerotle plaque in rabbit. Also we elucidate the impact of rosuvatation on the stability and expression of matrix matalloproteinase-2, matrix matalloproteinase-9,CD147in atherosclerotle plaque of rabbit.Methods1. Establishing the animal model18healthy male New Zealand white rabbits were selected.We randomly divided them into three groups:vacuity contrast group (n=6), model control group (n=6) and drug intervention group (n=6).Rabbits in model control group and idrug intervention group underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries after7days adaptive feeding with regular chow, then they were given a high-fat diet for threeteen weeks.At the end of14weeks,they were underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries,and after that give them7days adaptive feeding with regular chow. All rabbits were killed by an overdose of intravenous pentobarbital sodium,and the right carotid arteries were quickly removed for next expriment.Rabbits in vacuity contrast group weregiven a ordinary diet during the whole experimental process.2. Rabbits in the drug intervention group were randomized to receive gavage witn10mg/kg.d rosuvastain dissolved in10ml distilled water for14weeks after they were underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries.The others in acuity contrast group and model control group were randomized to receive gavage with10ml/day distilled water instead after they were underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries.3. Blood samples were collected at the beginning of the experiment and at the end of the experiment. The expression of matrix matalloproteinase-2and matrix matalloproteinase-9were detected by enzyme-linked immunosorbent assay (ELISA) as well.4.All rabbits were killed by an overdose of intravenous pentobarbital sodium at hen end of15weeks, the right carotid arteries were quickly removed for HE staining, Masson staining and Elastic tissue staining, and morphological characteristics of the plaque were observed under light microscopy. In order to identify the expression of inflammatory Factors and matrix metalloproteinase system in the plaque, monoclonal antibodies against,MMP-2,MMP-9were employed for immunohistochemical staining of the carotid arteries. The CD147mRNA and protein expression were respectively detected by RT-PCR and western blot by using the right carotid arteries.4. Statistic analysis SPSS13.0was applied for description and analysis of the data. Measurement data was represented in the form of (mean±Std deviation), paired samples t-test was used to compare means of two paired samples. Multiple means’ comparison adopted method of One-way ANOVA (Brown-Forsythe method was applied when equal variances not assumed). In cases those equal variances assumed, multiple comparisons of means adopted LSD test (Dunnett’sT3test was employed when equal variances not assumed).The significance level was0.05.Result1. The rabbits grew well generally. During the process of the test, noone rabbits died, all the18rabbits were used in result analysis. There are two rabbits in model control group suffered from cerebral infarction after underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries with a high-fat diet in the end of14weeks. All rabbits in the model control group had extensive sheets of elevated white-yellow plaques at the end of15weeks, the plaques were also more unstable,and had acutely ruptured plaque rupture-driven occlusive thrombus formation after triggering. Compared with the model control group, the plaques were stable in drug intervention group after triggeringin all the six rabbits at the end of15weeks.2. Variation of serum MMP-2concentration:The expression of MMP-2were detected by ELISA in the three group..The value of MMP-2in different groups have no significantly differents at the beginning of the experiment(P>0.05).At the end of the experiment,the value of MMP-2in the vacuity contrast group have no significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P>0.05). At the end of the experiment,the value of MMP-2in the model control group have significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P<0.05). the value of MMP-2in the model control group increased at the end of15weeks. At the end of the experiment,the value of MMP-2in drug intervention group have significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P<0.05).. the value of MMP-2drug intervention group increased at the end of15weeks.At the end of15weeks., the value of MMP-2in drug intervention group have significantly differents comapred with the model control group(P<0.05).MMP-2have a higher expression in model control group than drug intervention group. At the end of15weeks., the value of MMP-2protein in intervention group have significantly differents comapred with the vacuity contrast group (P<0.05).MMP-2protein have a higher expression in drug intervention group than vacuity contrast group.3. Variation of serum MMP-9concentration The expression of MMP-9were detected by ELISA in the three group..The value of MMP-9in different groups have no significantly differents at the beginning of the experiment(P>0.05).At the end of the experiment,the value of MMP-9in the vacuity contrast group have no significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P>0.05). At the end of the experiment,the value of MMP-9in the model control group have significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P<0.05). the value of MMP-9in the model control group increased at the end of15weeks. At the end of the experiment,the value of MMP-9in drug intervention group have significantly differents comapred with the value detected by ELISA at the beginning of the experiment(P<0.05).. the value of MMP-9drug intervention group increased at the end of15weeks.At the end of15weeks., the value of MMP-9in drug Intervention group have significantly differents comapred with the model control group(P<0.05).MMP-9have a higher expression in model control group than drug intervention group. At the end of15weeks., the value of MMP-9protein in intervention group have significantly differents comapred with the vacuity contrast group (P<0.05).MMP-9protein have a higher expression in drug intervention group than vacuity contrast group.4. CD147mRNA of each group detect by RT-PCR:There was CD147mRNA in each group at the end of15weeks.The value of CD147mRNA have significant differences during the three groups at the end of15weeks. At the end of15weeks., the value of CD147mRNA in intervention group have significantly differents comapred with the model control group(P<0.05). CD147mRNA have a higher expression in model control group than drug intervention group. At the end of15weeks., the value-of CD147mRNA in intervention group have significantly differents comapred with the vacuity contrast group (P<0.05). CD147mRNA have a higher expression in drug intervention group than vacuity contrast group.5. CD147protein of each group detect by western blot:There was CD147protein in each group at the end of15weeks.The value of CD147protein have significant differences during the three groups at the end of15weeks. At the end of15weeks., the value of CD147protein in intervention group have significantly differents comapred with the model control group(P<0.05).CD147protein have a higher expression in model control group than drug intervention group. At the end of15weeks., the value of CD147protein in intervention group have significantly differents comapred with the vacuity contrast group (P<0.05).CD147protein have a higher expression in drug intervention group than vacuity contrast group.Conclusion:The expression of CD147,MMP-2and MMP-9will significantly increase in unstable plaques and rosuvatation can stabilize the expression of CD147,MMP-2and MMP-9in the palques. Therefore,we assume that rosuvatation may play an important protective role in plaque stability of vulnerable plaque animal models by inhibiting the CD147,MMP-2and MMP-9expression.,and it could open a brand new wayfor the prevention and treatment of acute cardiovascular and cerebrovascular diseases.