The Effect Of Oxidized Modification On The Function Of High Density Lipoprotein And Its Relation With ABCA1

Background:Atherosclerosis (AS) is one of the important diseases that endanger human health nowadays. It can cause cardio - cerebral vascular diseases of which morbidity and mortality in the country and the world are very high. In recent yesrs, as experimental and clinical studies continuously, more and more attentions have been paid to lipids factors such as low density lipoprotein (LDL), high density lipoprotein (HDL), et al. These factors play a very important role in the pathologic physiologic process of cardiovascular diseases. Therefore there is necessity to find out the role of every factor in AS and their relations between eath other. The two key feature of AS are the degradation in plasma levels of HDL and the full filling of cholesterol in artery wall cells. Cellular cholesterol metabolism is very important in the pathogenesis of AS. The removal of cholesterol from the artery wall cells is a key step in the prevention of AS. Epidemiological studies have proven that high density lipoprotein cholesterol (HDL-C) can prevent the development of AS and serum levels of HDL-C is significantly negatively correlated with coronary heart disease, which is mainly attributable to the important role in the reverse cholesterol transport of HDL. HDL can remove cholesterol from the surrounding tissues (including atherosclerotic plaque) to the liver for recycling or excreting in the form of cholalic acid, a process known as reverse cholesterol transport (RCT). RCT can reduce the deposition of lipids in the vascular wall. A membrane protein called ATP-binding cassette transporter Al (ABCA1) mediated this metabolic pathway that excess cholesterol is transferred from cells to extracellular to from HDL. In 1999, the ABCA1 gene was found, and an upsurge was set off on the research of the characteristics of ABCA1. Many studies have found that ABCA1 play a role in the occurrence of AS: Wilcox et al. confirmed that ABCA1 not only mediated cholesterol efflux from cells, but also had a clear correlation with the formation of AS. ABCA1 gene expression was significantly higher in the arteriosclerosis region, and its injury and functional changes of endothelial cell is the important part of starting of AS. ABCA1 gene mutation may lead to Tangier disease and familial hypo-alpha-lipoproteinemia (FHA), which display as low HDL-C in patients and premature coronary heart disease. Currently, ABCA1 on the cell membrane of normal peripheral cells (monocytes / macrophages, endothelial cells, vascular smooth muscle cells, et al.) first mediate the outflow of phospholipids, and combinate it to apoA I to form the Phospholipid -apoA I complex outside cells. Cholesterol scatter through the cell membrane and then caught by discoid phospholipid-apoA I complex to form pre-HDL. Under the effect of lecithin cholesterol acyl transferase (LCAT), the globular mature HDL which rich in cholesterol ester is formed finally, and the process of RCT starts. In vivo, HDL is highly anti-AS. Lowering its standards has become an independent risk factor for coronary heart disease. However, in recent years, studies show that there is still oxidized high density lipoprotein (ox-HDL) in our body. Firstly Nakajima et al. detected the existence of ox-HDL in abdominal aortic atherosclerosis intima by using specific antibodies of ox-HDL which was oxidated by Cu²⁺ by chemical analysis. Subsequently, Nakano et al. also proved such a monoclonal antibody-based ELISA reaction can sensitively detect the plasma ox-HDL, reliably and specifically. Another study found that plasma with high endogenous triglyceride also has ox-HDL. In vitro experiments, when HDL exposed to the material that oxidated LDL, it could also be oxidated by Cu²⁺, hypochlorite and other oxidants, as well as endothelial cells, macrophages, and vascular smooth muscle cells. The more and more research is put on ox-HDL in recent years, and it has achieved a new understanding. They have become the focus of the studies today, What is the oxidation mechanism of HDL in vivo, which structure change happens after the oxidation of HDL, what kind of role it play in the process of AS, and how to prevent the oxidation of HDL, but the studies are not so much. ABCA1 taking an important role in RCT provides a new research direction in lipid metabolism and atherosclerosis. The further study of ox-HDL is of vital significance in understanding of human lipid metabolism and AS. It will make the treatment of dyslipidemia and AS to a new level.Objective:In this experiment, ECV-304 cells cultured in vitro were intervented by different concentrations of HDL, ox-HDL, anti-ABCA1. The expression of ABCA1 of ECV-304 cells, the cholesterol and its outflow, and the combination of HDL, ox-HDL and ECV-304 cells, were investigated to understand the effect of oxidized modification on the function of high density lipoprotein and its relation with ABCA1, explored that:1. ABCA1 expression of the endothelial cell(ECV-304 cells);2. Oxidative modification of high-density lipoprotein in the ABCA1-mediated cholesterol efflux activity of endothelial cells;3. The impact of oxidized modification on the combination of High Density Lipoprotein and endothelial cells and its relation with ABCA1.Methods:1. Separation, oxidation, lebeling with DiI and identification of human serum HDL: HDL was separated from human serum by one-step density gradient ultracentrifugation(10℃, 50 000 r/min, 5 h), oxidized by the Cu²⁺ (37℃, 50μmol/L, 24h), and labeled by the fluorescent probe DiI (37℃, 15mg/ml, 15h). Agarose Gel Electrophoresis and Bradford Protein Quantization and MDA Detection were used to identificate the product.2. ECV-304 cells cultured in vitro were observed under the electron microscope.3. ABCA1 expression of ECV-304 cells: by adding 1:100 anti-ABCA1 and 1:200 second FITC-labeled rabbit anti-sheep, ECV-304 cells were observed under fluorescence microscope.4. The effect of HDL and ox-HDL on the function of cellular cholesterol efflux of ECV-304 cells: after dealing with ox-LDL and cholesterol, the ECV - 304 cells were divided into five groups: A control group, B HDL group, C ox-HDL group, D anti-ABCA1 + HDL group, E anti-ABCAl+ox-HDL group. With or without joining HDL, ox-HDL or anti-ABCA1, ECV-304 cells were drumed dyeing by Oil Red O and hematoxylin after incubating for 24 h. Under the microscope, cells whose lipid droplets in the area equal to or greater than the area of nuclear were Oil Red O staining cells. Every piece of glass cell count 100. One-way ANOVA was used in multiple groups comparision.5. The combination of HDL or ox-HDL with endothelial cells: HDL and ox-HDL were labeled by the fluorescent probe DiI, as DiI- HDL and DiI-ox-HDL. ECV-304 cells slided on the coverslip were divided into five groups: A control group, B DiI-HDL group, C DiI-ox-HDL group, D anti-ABCA1+DiI-HDL group, E anti-ABCA1 +DiI-ox-HDL group. The combination of HDL or ox-HDL with endothelial cells were observed under fluorescence microscope with or without HDL, ox-HDL or anti-ABCA1.Results: 1. Separation, oxidation, lebeling with DiI and identification of human serum HDL: HDL was separated in 5 hours, and oxidized, labeled next. Agarose Gel Electrophoresis and Bradford Protein Quantization and MDA Detection showed that the HDL and ox-HDL were purified and the density was high. DiI-HDL could bind with ECV-304 cells. The results proved that the DiI-HDL had the normal lipoprotein ligandbinding properties.2. ECV-304 cells grew well with eumorphism.3. Red fluorescent material was observed on vascular endothelial cells under fluorescence microscope.4. The effect of HDL and ox-HDL on the function of cellular cholesterol efflux of ECV-304 cells: the number of Oil Red 0 staining cells of A, B, C, D, E groups were 45±6, 27±6, 51±8, 53±6, 61±4 (per 100 cells). Compared with group A, Oil Red 0 staining cells of group B decreased significantly (P = 0. 000), and Oil Red O staining cells of group C did not decrease or increase significantly (P = 0. 129), and Oil Red O staining cells of group D and group E increased significantly (P = 0. 031, P = 0.000). Compared with group B, Oil Red O staining cells of group D increased significantly (P = 0. 000), and Oil Red O staining cells of group D increased significantly (P = 0. 000). Compared with group C, Oil Red O staining cells of group Group E increased significantly (P = 0. 010). It shows that ECV-304 cells reduced cholesterol efflux significantly in Anti-ABCA1 conditions, and that HDL promoted cholesterol efflux and that ox-HDL could not promote cholesterol efflux without Anti-ABCA1.5. The combination of HDL, ox-HDL and endothelial cells: compared with 0.01mol/L PBS control group, the DiI- HDL and the DiI-ox-HDL were both binding with ECV-304 cells with or without the addition of anti-ABCA1. But compared with DiI- HDL, the binding with ECV-304 cells of DiI-ox-HDL was significantly weaken. Conclusions:1. This method is simple, rapid and reliable. It could be applied in lipoprotein receptor study successfully.2. Vascular endothelial cells(ECV-304 cells) can express ABCA1.3. The RCT of endothelial cells was mediated by ABCA1, but anti-ABCA1 could block RCT. After oxidative modification, the structure of HDL changes, which had affected the function of RCT of endothelial cells.4. The oxidized modification of HDL induced the structural change, which affected the binding with the vascular endothelial cells. Between HDL and ox-HDL in binding with the ECV-304 cells, there was not significant relationship with ABCA1.