The Affect Of TF On The Expression Of MCP-1in Human Umbilical Vein Endothelial Cells

BackgroundAtherosclerosis(AS) is a pathological remodeling process in the systemic arterial wall. It forms the basis for most cardiovascular and cerebrovascular diseases. Endothelial cell injury is an important initial event at the onset of atherosclerosis. Endothelial cell may synthesize the monocyte chemotactic protein (MCP) under given conditions, such as stimulation with Ox-LDL. Chemotaxis of monocytes combines with endothelial cell receptors by monocyte surface molecules. Subsequently, monocytes immigrat into the inner lining of an artery under monocyte chemotactic protein and other chemotactic factors, and then differentiate into macrophages. Due to their role in phagocytosing lipid, macrophages transform into foam cells (macrophages full of lipid material). This is not only a sign of fatty-streak formation, but also an evidence of beginning of atherosclerosis. Therefore, mononuclear phagocyte system plays an important role in the formation of atherosclerosis.Macrophages in fatty streak plaques have many changes in functional. AS plaque in situ hybridization analysis showed that macrophages containing intracellular coding, tissue factor and apolipoprotein E, transforming growth factor-beta and tumor necrosis factor messenger RNA. Macrophage-produced Cytokines can be used as protein medium of inflammatory and immune responses transmit the information between.vascular endothelial cells and smooth muscle cells by the way of autocrine or paracrine. However, many functions of macrophages is regulated by the monocyte/macrophage chemotactic factor in the lesion area. Accordingly, the monocyte/macrophage chemotactic factor and its control of macrophage function significantly affects the occurrence and development of AS.Chemokines can be divided into four subfamilies:C-C, C-X-C, C and CX according to the structure, function and genetic characteristics. And C-C subfamily is close to monocyte/macrophage, including MCP-1-5, MIP-1, RANTES, the monocyte chemotactic protein-1(MCP-1) plays an important role in atherosclerosis pathological processes.MCP-1is a single-chain protein consisting of76amino acid residues and one of the monocyte/macrophage chemotactic proteins. Three main cell types present in atherosclerotic lesions, including endothelial cells, smooth muscle cells (SMCs) and mononuclear cells, are closely related to MCP-1.MCP-1specifically acts on the blood monocytes, attracts them move into the subendothelial space and constitutes the important mechanisms of pathogenesis of atherosclerosis. MCP-1is involved in macrophages’chemotaxis and migration toward atherosclerotic plaque and enhanced the immune inflammatory reaction. It can induce tissue factor (Tissue factor, TF) expression and plays an important role in the atherosclerotic plaque and thrombus formation.When MCP-1induces increased expression of TF in inflammatory cells such as monocyte, it may lead to disastrous results. Because TF is the only cell surface expression of transmembrane glycoprotein in blood coagulation system and one of the most powerful coagulant in vivo. Foreign scholars speculate that coagulation and inflammation mediated by TF can promote each other to form the Coagulation-Inflammation-Thrombosis circuit, which may play a critical role in the pathogenesis of coronary heart disease (CHD). AS is the important pathogenetic basis of CHD, and TF plays an important role in Coagulation-Inflammation-Thrombosis circuit result in CHD. The further researches on TF in the mechanism of AS pathological process have great significance for study of revealing the pathogenesis of CHD. The domestic and foreign researches show that the vascular endothelial cells, monocytes and smooth muscle cells can generate TF. Moreover,TF can promote cell proliferation of vascular smooth muscle cells and endothelial cell apoptosis in the occurrence and development of atherosclerotic lesions. These findings clearly illustrate that there are some interactions between TF and the cells content in atherosclerotic plaques. Mononuclear cells as one of the major cells in atherosclerotic plaques, therefore, it is very important to research its interaction with TF,which will be to further reveal the pathogenesis of CHD.It is known that MCP-1plays a vital role in monocyte/macrophage chemotaxis and aggregation. MCP-1is involved in macrophages’chemotaxis and migration toward atherosclerotic plaque and enhanced the immune inflammatory reaction. It can induce tissue factor (Tissue factor, TF) expression and also plays an important role in the atherosclerotic plaque and thrombus formation. MCP-1and TF expression might reflect the stability of atherosclerotic plaques. Consequently, to study the expression of MCP-1and TF in plasma of patients with coronary heart disease and the relationship between MCP-1and TF has important significances in the further study, Such as the interaction between monocytes and TF, and more profoundly revealed TF and MCP-1in pathologic process of AS and Coagulation-Inflammation-Thrombosis circuit.Chapter one:The Significance of plasma MCP-1、TF in patients with Coronary Heart DiseaseObjective:1. To investigate the significance and clinical value of plasma MCP-1and TF in CHD by observation of the concentrations of plasma MCP-1and TF in patients.2. To investigate the relationship between MCP-1and TF in patients’plasma.Methods:122cases of patients had been admitted to the Cardiology department of Southern Medical were enrolled between January2010and June2011.Exclusion criteria in this study were patients with history of heart failure, pulmonary infarction, pericarditis, myocarditis, cardiomyopathy, aortic dissection, peripheral vascular disease, cerebrovascular disease, acute and chronic inflammation, immune and connective tissue diseases, fever, hyperthyroidism, secondary anemia caused by angina pectoris symptoms. All patients were divided into NCHD group (n=30)、SAP group (n=32)、UAP group (n=27)、AMI group (n=33). Relevant examinations were done after admission. In all these groups, the concentrations of plasma MCP-1and TF in the patients was detected by ELLSA.The SPSS13.0analysis software was used to analyze date. Data was expressed using mean±standard deviation. Independent t-test was used to test the difference between the two groups. One-Way ANOVA was used to test the difference in multiple groups (The method of LSD or Bonferroni was used for multiple comparisons in variance homogeneity. The method of Welch or Brown-Forsythe was used for variance nonhomogeneity and the method of Dunnett T3or Dunnett C was used for multiple comparisons).The Partial was used to judge the relationship between two variables. P<0.05was considered significant difference.Results:1. The concentrations of plasma MCP-1, TF of NCHD group,SAP group,UAP group,AMI group all have significant differences (all P<0.05);2. The concentrations of plasma MCP-1TF, cTnI,CK-MB in the patients between CHD and NCHD groups have significant differences (all P<0.05);3. There are positive correlation between MCP-1and TF in patient plasma,the correlation coefficient is0.278(P=0.012, P<0.05)Conclusion:1. Plasma MCP-1、TF may have clinical significance in CHD.2. The concentrations of plasma TF in Patients was correlated with MCP-1. It shows that MCP-1may play an important role in Coagulation-Inflammation-Thrombosis circuit. Chapter two:The affect of tissue factor on the expression of monocyte chemotactic protein-1in human umbilical Vein endothelial cellsObjective:By Observation the effects of different concentrations of tissue factor (TF) on the expression of monocyte chemotactic protein-1(MCP-1) in human umbilical vein endothelial cells, to study the corresponding mechanisms of TF on the role of MCP-1.Methods:Human umbilical vein endothelial cells were cultured in vitro and divided into control group and10ng/L,100ng/L,103ng/L,104ng/L of tissue factor treatment groups. Sterile water was add to the control group, the corresponding concentration of tissue factor were respectively added to the treatment groups. Using ELLSA and RT-PCR methods detect the expression of MCP-1and MCP-1mRNA in human umbilical vein endothelial cells respectively. The SPSS13.0analysis software was used to analyze date. Data was expressed using mean±standard deviation. One-Way ANOVA was used to test the difference in multiple groups (The method of ANOVA was used for variance homogeneity and the method of LSD or Bonferroni was used for multiple comparisons. The method of Welch or Brown-Forsythe was used for variance nonhomogeneity and the method of Dunnett T3or Dunnett C was used for multiple comparisons). P<0.05was considered significant difference.Results:1.The ELLSA results showed:there was expression of MCP-1in human umbilical vein endothelial cells of all groups. The expression level of MCP-1of the control group comparing to those of four tissue factor treatment groups all have significant differences (all P<0.05). Among the four tissue factor treatment groups, the expression of MCP-1in human umbilical vein endothelial cells has significant differences (all P<0.05), within a certain concentration range. The TF promote the expression of MCP-1in human umbilical vein endothelial cells with Concentration-dependent.2. The RT-PCR results showed:there were expression of MCP-1mRNA in human umbilical vein endothelial cells of all groups. The expression level of MCP-1mRNA of the control group comparing to those of four tissue factor treatment groups all have significant differences (all P<0.05). Among the four tissue factor treatment groups, the expression of MCP-1mRNA in human umbilical vein endothelial cells has significant differences (all P<0.05), within a certain concentration range. The TF promote the expression of MCP-1mRNA in human umbilical vein endothelial cells with Concentration-dependent.Conclusion:Tissue factor can promote the expression of monocyte chemotactic protein-1in human umbilical vein endothelial cells and with Concentration-dependent. Chapter three:The affect of tissue factor on Human umbilical vein endothelial cells-monocytes adhesion reactionObjective:By Observation the effects of different concentrations of tissue factor (TF) on Human umbilical vein endothelial cells-monocytes adhesion reaction, to explore the mechanism of TF in the pathological process of atherosclerosis.Methods:Human umbilical vein endothelial cells were cultured in vitro and divided into the without TF group, monocytes group and10ng/L,100ng/L,103ng/L,104ng/L of tissue factor treatment groups.The monocytes group without human umbilical endothelial cells. Add monocytes and the corresponding concentration of tissue factor. Microscope were used to observe the effect of endothelial cell-monocyte cell adhesion after incubation for12hours at37℃,and then Cells were washed with PBS and centrifugalized three times,washing away not adherence of mononuclear cells. in a6mol/L sodium hydroxide solution dissolves the cell at room temperature. The protein content of cells lysate was observed by Lowry method. The adhesive ratio of endothelial cell-monocyte cell was calculated according to the formula:adhesive ratio (%) The SPSS13.0analysis software was used to analyze dates. Data was expressed using mean±standard deviation. One-Way ANOVA was used to test the difference in multiple groups (The method of ANOVA was used for variance homogeneity and the method of LSD or Bonferroni was used for multiple comparisons. The method of Welch or Brown-Forsythe was used for variance nonhomogeneity and the method of Dunnett T3or Dunnett C was used for multiple comparisons). P<0.05was considered significant difference.Results:Monocytes adhesion to human umbilical vein endothelial cells in the tissue factor treatment groups. There are significant differences in the adhesion rate of endothelial cell-monocyte among tissue factor treatment groups (P<0.05). Within a certain concentration range,the tissue factor of the culture medium promote adhesion rate of endothelial cell-monocyte in concentration-dependent.Conclusion:Within a certain concentration range,tissue factor promote adhesion rate of vitro cultured human umbilical vein endothelial cells-peripheral blood mononuclear cells in concentration-dependent It may play an important role in the atherosclerotic disease process.