Selection Of Reference Genes And The Mechanisms Of ADAMTSs Expression In Myocardial Infarction

Background and objectiveThe importance of gene expression analysis in many areas of life science research is growing. Its in-depth study will benefit to explore disease-related genes, understand gene regulation, resolve the mysteries of life, and ultimately to service to humanity. Real-time fluorescence reverse transcription-polymerase chain reaction is the most sensitive method of studying specific messenger RNA quantitatively. To analyze the differences of the specific messenger RNA content, the internal reference gene is used to quantify. There are hundreds of housekeeping genes. At present, the most widely used internal reference housekeeping genes are GAPDH, β-actin,18SrRNA and28SrRNA. Using of one housekeeping gene singly or selection housekeeping genes blindly in accordance with different experimental subjects may make the small differences in gene expression difficult to find, on the other hand it may lead to errors or even opposite conclusions. Rat is a commonly used animal to be made myocardial infarction model. It is the first choice for many laboratories to use rats as animal model for coronary heart disease-related genes studies. However, there are fewer studies about the selection of reference genes in rats after myocardial infarction.Myocardial infarction is a major disease to threat human life. Animal model of myocardial infarction is significant greatly for studying pathogenesis and pathophysiology of human coronary heart disease and evaluation of treatment methods. The method of coronary artery ligation is used to prepare the model ever since a long time ago. In practice, many reasons result in animal death or making model failure just as anesthesia, using respirator inadequately, lung injury and the left coronary artery positioning erroneously. Therefore, it is a core issue of improving survival and the success rate of making model to solve the question of animal model of myocardial infarction. We consummate the method of making rat model of myocardial infarction and improve the survival rate of rats significantly.Acute myocardial infarction is a leading cause of death and disability in world. Ventricular remodeling after myocardial infarction can lead to heart failure and mortality. The dynamic change of extracellular matrix (ECM) accumulation and degradation is included in the process of ventricular remodeling. Many biological substances such as proteases, protease inhibitors and growth factors play a part in ECM reconstruction. The increased expression and activation of matrix metalloproteinases (MMPS) is also involved. In addition to matrix metalloproteinases, there are other protease families having the capacity to degrade the ECM. ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) is a group of secreted proteases that now incorporates19genes in humans, involving of cracking a variety of proteoglycans, collagen metabolism, anti-angiogenesis, VWF polymer degradation, the embryonic organ development, reproduction and other functions. Some of ADAMTS can bind to the ECM. ADAMTS4and8have been shown inflammatory regulated enzymes expressed in macrophage-rich areas of carotid atherosclerosis plaque and coronary instable plaque. ADAMTS4expression increases in the development of atherosclerosis process. Versican is a member of the aggrecan family, spread in the organization widely and participate in the process of wound healing and tissue remodeling. Versican expressed and incrased temporally in the myocardium of coronary artery ligation-induced myocardial infarction rat model, suggesting their involvement in the inflammatory response after myocardial infarction. ADAMTS4degrade versican of the vessel wall at V1/V0versican's Glu1428-Ala1429site. ADAMTS4can be blocked by the endogenous inhibitors TIMPs. ADAMTS4and ADAMTS5can be effectively suppressed by TIMP-3, while not be sensitive to TIMP-1,2and4.In recent years the role of ADAMTS in inflammation and atherosclerosis is attentioned. Immunohistochemical analysis showed that ADAMTS1,4,5and8expressed in the human carotid artery plaque and coronary atherosclerotic plaque. ADAMTS4,5and8co-exist with macrophages and ADAMTS1co-exist with smooth muscle cells and endothelial cells. Versican is widely distributed in the organization and also present in the heart. Versican expression increased in the myocardial infarction heart and came from the mononuclear cells, suggesting their participation in the inflammatory response after myocardial infarction. Aggrecan is a cartilage-specific protein of chondroitin sulfate polysaccharide and is a substrate of ADAMTS. ADAMTS4and5showed strong activity in the degradation of aggrecan. It is not clear about the expression position of ADAMTS4in the heart after myocardial infarction. Whether versican express in the same area of ADAMTS4and whether aggrecan express in the heart after myocardial infarction are unclear. The shear stress up regulated the ADAMTS1mRNA expression in the human vein endothelial cells and cardiac microvascular endothelial cells. In the atherosclerotic plaque intima and proliferation/migration vascular smooth muscle cells, ADAMTS1mRNA expression increased. Stimulation macrophages with INF-γ, TNF-α and IL-1γ showed ADAMTS4,7,8,9expression increased, ADAMTS1and17expression decreased, while ADAMTS2,5,10is not affected after stimulation with INF-γ. ADAMTS4,7and8expressions increased, ADAMTS9slightly elevated after stimulation with TNF-α. ADAMTS4,7and8expressions were not affected, ADAMTS1and9increased in the early phase after stimulation with IL-1β. All of these indicate there are different expression and regulation mechanisms of ADAMTS family members. Previous observations suggest that (a) ADAMTS4plays an important role in the development of atherosclerosis and instability of atherosclerotic plaques;(b) ADAMTS4may serve as a marker of plaque destabilization and for predicting the severity of ACS. Percutaneous coronary intervention (PCI) procedure can be regarded as a model for mechanical induced plaque rupture. Thus studies on the inflammatory reaction during PCI could give information on mechanisms of plaque destabilization. Many clinical studies have evaluated the inflammatory response after PCI in patients with CAD. Since ADAMTS4has been proved to be inflammatory regulated enzymes and involved in the development of atherosclerosis and instability of atherosclerotic plaques, we speculated ADAMTS4level would elevate in the coronary circulation of patients with CAD and be influenced by PCI.In this study, we compared four housekeeping genes'expression in hearts of myocardial infarction rats with real-time fluorescence reverse transcription-polymerase chain reaction method and analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats. The dynamic expression changes and distribution area of ADAMTS4, ADAMTS8, versican, TIMP3and the possible mechanisms were studied. The changes and interactions of local ECM molecules after acute myocardial infarction were further understanded and a new theoretical basis for treatment was provided. ADAMTSs expression trend in the endothelial cells after stimulation withlL-1β were detected. We detected coronary ADAMTS4and high-sensitivity C-reactive levels using coronary sinus sampling and assessed the effect of PCI on ADAMTS4and hs-CRP levels. The main research contents and results are as follows:1. Selection of housekeeping genes in rat model of myocardial infarction We made myocardial infarction models using the anterior descending coronary artery ligation method,combined with practical work, analyzed and selected the four widely used standard housekeeping genes:glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal protein L13A (RPL13A), beta-actin (ACTB) and acidic ribosomal phosphoprotein PO (ARBP). We compared their expression in the rat heart after myocardial infarction using real-time fluorescent reverse transcription polymerase chain reaction, analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. The results showed the M values of RPL13A, GAPDH, ARBP and ACTB gene were0.812,0.721,0.812and1.2respectively. The results suggest that GAPDH and ARBP are the most stable genes for the rat myocardial infarction model.2. Improvement of the rat myocardial infarction modelOne hundred male Wistar rats (weight230-270g) were divided into model group (n=80) and sham-operatered group (n=20). The rats were anaesthetised with sodium pentobarbital (40mg/kg, intraperitoneally), operated tracheostomy, then ventilated with an automatic breathing apparatus using the volume control mode(tidal volume,3ml/100g, respiratory rate,60-70cycles/min, respiratory ratio,1:1). An left anterior thoracotomy was performed at the fourth intercostal space and rib was retracted with the mastoid retractor for exposing the heart. The left anterior descending coronary artery was ligated approximately3mm from its origin with the use of6-0silk to induce MI. The induction of MI was confirmed by a cardiac surface color change from reddish to a pale color and by ST-segment elevation documented by continuous electrocardiographic monitoring or left ventricular motion weakened. Sham-operated rats underwent similar surgery but without coronary artery ligation. Then closed thoracic cavity layer by layer, removed ventilator and tracheal intubation. To prevent the airway stegnosis and secretions lead to suffocation, the trachea and the neck incision were not closed. Warmed the rats with40W light bulb12h after surgery, observed the airway secretions4h after surgery and removed with suction tube in time. The results showed the success rate of making myocardial infarction model was98.6%, the survival rate of model group3weeks after surgery was88.75%and survival rate of sham-operated group was100%. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats.3. The dynamic expression of ADAMTS4, ADAMTS8, versican and TIMP-3genes in rat myocardium after myocardial infarctionChose250-300g male Wistar rats and made myocardial infarction model by the method mentioned above. pats were sacrificed anesthesia overdose at3h,6h,12h,24h,3days,7days,14days, or21days (n=5) after surgery and removed the heart rapidly. The infarcted myocardium below the ligature points were separated and put into the-70℃refrigerator. The ADAMTS4, ADAMTS8, versican and TIMP-3mRNA expression were analyzed by real-time fluorescence quantitative RT-PCR. ADAMTS4protein expression was analyzed by ELISA assay. The results showed ADAMTS4mRNA expression was significantly increased in the infarcted hearts6h and12h after MI compared with the level in sham-operated hearts (p<0.05), then decreased rapidly. While the relative level of ADAMTS8increased at6h, peaked at24h, remained high at3days(p<0.05),then decreased gradually. After myocardial infarction versican mRNA levels were significantly increased, peaked at3days and remain high for a long time. TIMP3expression levels decreased. ADAMTS4protein levels was significantly increased at6hours (p=0.026),12hours (p=0.003),24hours (p=0.002) and3days (p=0.009) after myocardial infarction.ADAMTS4, ADAMTS8, versican, and TIMP-3mRNA expressed in rat heart after myocardial infarction and presented different dynamic trends. There are interrelationship among the expression of ADAMTS4, versican, and TIMP-3.4. ADAMTS4expressed in endothelial cells and myocardial cells of the rat infarcted heartMale Wistar rats (250-300g) were chosed to make myocardial infarction model. Rats were killed3days after surgery and the left ventricle were taken to produce frozen section with slice thickness5um. ADAMTS4, versican and aggrecan protein expression were detected by immunohistochemistry method. For the negative control group, we use the same dilution of rabbit IgG to handle slices. The results showed ADAMTS4expressed in myocardial cells at marginal zone of the infarcted area and did not express at normal myocardium and area away from infracted myocardium. At marginal zone of the infarcted area ADAMTS4expressed weakly and versican had strong expression in capillary endothelial cells. Aggrecan showed no expression in the cardiac tissue and vascular endothelium of MI group and sham-operation group. ADAMTS4and versican expression co-existed in endothelial cells hinted a link between them. The dynamic change may play a role in matrix remodeling after myocardial infarction.5. The expression of ADAMTSs in endothelial cells stimulated with interleukin-1βSelected human umbilical vein endothelial cells to subculture. Stimulated endothelial cells with2.5ng/ml,5ng/ml,10ng/ml,20ng/ml IL-1β respectively (n=5) and collected cells to extract total RNA and protein24hours after stimulation. Stimulated endothelial cells with10ng/ml IL-1β and collected cells to extract total RNA and protein respectively2h,6h,12h,24h,48h (n=5) after stimulation. Blank control group were set up. ADAMTS1,4,8,9,15mRNA expression were analyzed by RT-PCR and protein expression by Western-blot. The results showed after stimulation with IL-1β ADAMTS1,4,8,9,15mRNA expression were all increased, but the trends with time was different, ADAMTS4protein expression increased.The results suggest that ADAMTSs involved in inflammation, the specific mechanism remains to be further clarified. 6. The change of coronary level of ADAMTS4and effect of percutaneous coronary intervention on ADAMTS4in patients with coronary artery diseaseThe81subjects were classified according to their coronary angiographic findings into control, simple, and complex groups. PCI was performed in35patients. Blood samples were taken from the coronary sinus. ADAMTS4and hs-CRP levels were measured by ELISA and turbidometry respectively. Results:ADAMTS4and hs-CRP showed higher level in complex group than in control and simple group (P<0.001). The ADAMTS4level showed a positive correlation with the hs-CRP level whether from all patients or from patients with CAD (r1=0.73, r2=0.763, P<0.01). The ADAMTS4value was higher in patients who underwent PCI than in those who did not (P<0.001), and the same was found regarding CRP (P=0.025).Coronary ADAMTS4and hs-CRP levels elevated in patients with CAD and increased with the complexity of the lesions. The marked increase of ADAMTS4and hs-CRP after PCI in patients with CAD can be attributed to their release from the coronary atheroma secondary to the direct mechanical effect applied on the atheroma itself by balloon inflation and stent deployment.