Peripheral Cell Injury In Hypertensive Patients

Study Objectives:The overall object was divided3aspects:firstly, it was to establish the ways of isolation, preparation, investigation and detection of immunophenotypes of plasma endothelial microparticles (diameter was from0.5um to lum, diameter was less than0.5um).Secondly, it was to investigate different immunophenotypes and characters of endothelial microparticles were from the peripheral blood of patients. Thirdly, it was to analyse the data of patients by multiple statistical analysis method, including univariate analysis and multivariate analysis. We discussed endothelial microparticle as a biomarker of vascular disease and the pathophysiological mechanisms of endothelial dysfunction.The objective of first part:To analyse immunophenotypes, related factors and diagnostic threshold by investigating different phenotypes of endothelial microparticles and the number and percentage of endothelial microparticles in different levels of hypertension patients (including middle-uncontrolled hypertension, sever-uncontrolled hypertension and well-controlled hypertension) and healthy people. We also discussed the pathophysiological mechanisms, endothelial microparticle was potential to be biomarkers to diagnose and monitor endothelial dysfunction.The objective of second part:To find the distinguish phenotype of endothelial microparticles,it could further lead to endothelial dysfunction and was associated the development of coronary artery disease, we compared with the number and percentage of endothelial microparticles in hypertension without coronary artery disease and hypertension with coronary artery disease. We also analysed the immunophenotypes, associated factors and diagnostical threshold.The objective of third part:To find the special immunophenotype of small endothelial microparticle in hypertension patients, we investigated the number and percentage of endothelail microparticles in the peripheral blood of hypertension patients and healthy people. We also discussed the potential vaule of small endothelial microparticle to monitor endothelial dysfunction in hypertension patients.Study methods:The overall research method applied for flow cytometry technique and multiple statistical analysis method.First part and second part:Blood samples were drawn by venipuncture into blue-top vacutainer tubes containing sodium citrate. Whole blood (3ml) of each sample (including hypertension patients, hypertension patients with coronary artery disease and healthy people) and was centrifuged to obtain platelet-poor plasma within lhour of blood sampling, and stored at-80℃until analysis. When tested the endothelial microparticle, the specific antibody monoclonal antibodies were added at room temperature. For flow cytometry assay, events calibrated by an internal standard beads(0.8um;Sigma,St.Louis,MO) was applied for determining the size of microparticle,the number and percentage of endothelial microparticles were tested by flow cytometery.EMP was defined as CD31-positive and CD42-negative, or CD62E-positive or CD144-positive or CD51-positive particles. In order to study the relationship between endothelial microparticle (EMP) and platelet microparticle (PMP),we also tested the number and percentage of PMP. PMP was defined as CD31-positive and CD42-positive or CD62P-positive particles. Spearman correlation and line regression was applied to analyse the correlation among MPs and between MPs and clinical parameters.ROC curve was applied to assess the diagnostic value of MPs. The number and percentage of EMP and PMP were compared by statistics.Third part:The3ml blood was drawn from each sample(hypertension patients and healthy people), and was centrifuged to obtain platelet-poor plasma within lhour of blood sampling and stored at-80℃until analysis.When tested the endothelial microparticles, the specific antibody monoclonal antibodies were added at room temperature.0.46um internal standard bead was used to determine the size of endothelial microparticle and the threshold was set up at35,000.The number and percentage of CD31+/CD42b" EMP and CD62E+EMP was detected and compared by statistics.Study results:First part:The number of CD31+/CD42b-EMP was higher in different hypertension groups(middle-uncontrolled hypertension, severe-uncontrolled hypertension and well-controlled hypertension) than in healthy group(both P<0.01).We did not find statistical significance in hypertension groups. The number of CD51+EMP and CD42b+PMP was lower in well-controlled hypertension than in healthy group(both p<0.05).The number of CD62E+was both higher in middle-uncontrolled hypertension and well-controlled hypertension than in healthy group(both p<0.05).We did not find statistical significance of other immunophenotypes of EMP. The percentage of CD31+/CD42b"ENP、CD144+EMPand CD62E+in different hypertension groups was higher than in healthy group. It is noticeable that the percentage of CD62E+EMP was higher in well-controlled hypertension than in middle-uncontrolled hypertension and severe-uncontrolled hypertension. The percentage of other MP was not statistical significance among groups.Second part:The number of CD31+/CD42b-was higher in different hypertension groups(hypertension with coronary artery disease and hypertension without coronary artery disease)than in healthy group(both P<0.01).The number of CD62E+EMP was higher in hypertension with coronary artery disease than in healthy group(P=0.014).The percentage of CD31+/CD42b-EMP, CD144+EMP and CD62E+EMP was higher in different hypertension groups than in healthy group(both p<0.01).Third part:The percentage of CD62E+small endothelial microparticle was higher than in healthy group(P<0.01),the area under ROC curve of percentage of ROC was0.865,the best diagnostical threshold was1.15.Study conclusionsThe phenotypes of CD31+/CD42b-、CD144-、CD62E+EMP showed high potential value to monitor endothelial dysfunction in hypertension patients,our experiment also provided a new pointcut to study hypertension. The CD62E+EMP may judge the prognosis in hypertension and hypertension with coronary artery disease. The CD31+/CD42b+and CD62P+PMPshowed synergistic effect with endothelial microparticle, including monitor, judgement of prognosis and pathological and physiology function.Limitations1.The samples of experiment were not big enough,it needs more studies to support the results.2The age of patients and healthy subjects was not matched enough,although we found age was not correlated with microparticles by multiple line regression,we also need to do experiements to support our results under the age well-matched.3.It also needs studies to study the effects of medicineAIt needs studies to investigate the mechanisms how microparticles cause to endothelial dysfunction.Further research work:It needs to study the generation mechanisms of different phenotypes of EMPs. It needs to study how the EMPs cause to endothelial dysfunction and the pathophysiology mechanism of hypertension development.