Study On The Value Of Endothelial Progenitor Cells In The Evaluation Of Nondisabling Ischemic Cerebrovascular Events

PARTⅠ FUNCTIONAL CHANGES OF ENDOTHELIAL PROGENITOR CELLS OF PATIENTS IN NONDISABLINGISCHEMIC CEREBROVASCULAR EVENTSBackground:TIA and minor stroke were contained in NICE,accounting for about40% of ischemic cerebrovascular events.The 90-day recurrence rate was20% for TIA and minor stroke,and nearly half of their patients had functional disability or death.Endothelial dysfunction is the cause of atherosclerosis,and atherosclerosis is a common cause of ischemic cerebrovascular disease.Although mature vascular endothelial cells contribute to endothelial repair,their regenerative capacity is limited.As a result,people are increasingly concerned with EPCs in vascular repair.EPCs are stem cells that can differentiate into vascular endothelial cells.The severity of cerebral infarction is directly related to the number ofcirculating EPCs,and severe functional disability is correlated with the decrease of circulating EPCs.Compared with the normal population,patients with atherosclerosis have fewer circulating EPCs and the EPCs have lower migration and proliferation capacity.Meanwhile,patients with less circulating EPCs are often accompanied by atherosclerosis.It is not difficult to understand that various atherosclerosis risk factors ultimately affect the number and function of EPCs,and the number and function of EPCs directly affect the repair ability of vascular endothelium.In the clinical work of neurology,patients with progressive stroke often encountered from TIA and minor stroke,so we believe that there may be differences in the function(proliferation,differentiation and migration ability)of circulating EPCs in NICE patients,which may lead to the development of cerebral infarction in some NICE patients.At present,the relationship between EPCs and NICE is rarely reported,so this study focused on the function of circulating EPCs in NICE patients.Objective:By comparing the adhesion,proliferation and migration ability of circulating EPCs in NICE group,ACI group and NC group,to investigate the influence of the heterogeneity of circulating EPCs on non-disabling ischemic cerebrovascular events.Methods:30 NICE patients within 24 hours after the onset were continuouslycollected.According to the assessment criteria for high-risk non-disabling ischemic cerebrovascular(HR-NICE)events,they were divided into the HR-NICE group and the NHR-NICE group.Meanwhile,15 patients with acute cerebral infarction(ACI)and 15 healthy volunteers during the same period were selected into ACI group and NC group.Density gradient centrifugation was used to isolate circulating EPCs.DIL-acLDL/FITC-UEA-lectin phagocytosis assay was used to identify EPCs,after 72 hours of culture,adopt MTT method to measure the proliferation of circulating EPCs in each group.Boyden chamber method was used to measure the migration ability of EPCs.Results:1.The cells obtained by density gradient centrifugation were scattered small round cells after 24 hours of culture.After 72 hours of culture,the cells became larger and colonies appeared,and the surrounding cells grew radially.Dil-acldl/ FITC-UEA-lectin double phagocytosis assay identified these cells as EPCs.2.Compared with the NC group,the adhesion ability of EPCs cells was weakened in the HR-NICE group,the NHR-NICE group and the ACI group,while the ACI group was the worst adhesion ability.Compared with the NHR-NICE group,the cell adhesion ability of the HR-NICE group was decreased(P<0.05).3.The EPCs migration ability showed that compared with the NC group,the HR-NICE group,the NHR-NICE group and ACI group had weaker migration ability,and the ACI group had the worst.Compared with the NHR-NICE group,the HR-NICE group had worse migration ability(P<0.05).4.The results of EPCs proliferation capacity showed that compared with the NC group,the proliferation ability of the HR-NICE group,the NHR-NICE group and the ACI group was decreased,and the ACI group was the worst.Compared with the NHR-NICE group,the proliferation ability of the HR-NICE group decreased(P<0.01).Conclusions:1.EPCs extracted by density gradient centrifugation EPCs were identified by morphology and phagocytosis assay.2.The decreased adhesion ability,proliferation ability and migration ability of EPCs may be one of the reasons why NICE developed into cerebral infarction.PART Ⅱ ENDOTHELIAL PROGENITOR CELLS IN THE EVALUATION OF NON-DISABLING ISCHEMIC CEREBROVASCULAR EVENTSBackground:NICE can relapse and get worse in a short period of time,which is called HR-NICE.HR-NICE included patients with minor stroke,high-risk TIA,acute multiple cerebral infarction,intracranial or extracranial large artery atherosclerosis with a stenosis rate greater than 50%.TIA and minor stroke have a higher risk of recurrence,some indicators have been developed to predict the risk of recurrence.These indicators included clinical predictors(e.g.,ABCD2,California risk assessment scale,etc.),imaging predictors(new infarct lesions and arterial stenosis rate)and biomarker predictors(e.g.,glycosylated albumin,hypersensitive c-reactive protein).It is worth concern that the biological predictors involved in the assessment tools are all indicators of vascular injury,and no biomarkers for vascular repair are proposed.After stroke,we should pay attention to vascular repair,because the ability of vascular repair is also one of the factors to evaluate the long-term prognosis of NICE.ABCD2 should be combined with other indicators to better predict the risk of stroke recurrence.EPCs can improve endothelial dysfunction and promote angiogenesis.The mobilization and homing of EPCs are promoted by some cytokines.Among them,SDF-1α has been the most studied,and SDF-1α/CXCR4 axis promotes the release,migration and homing of EPCs.Combined with EPCs and SDF-1 as factors to promote vascular repair,we suspected that there might be differences in the number of circulating EPCs and the serum SDF-1α level in NICE patients,which might lead to recurrent attacks or even cerebral infarction in some NICE patients,namely the HR-NICE population.EPCs and SDF-1α may be used as new biological indicators to predict relapse in NICE and as early risk assessment indicators for HR-NICE.Objective:To explore whether the number of circulating EPCs and serum SDF-1α concentration in NICE patients could be used as early risk assessment indicators for high-risk non-disabling ischemic cerebrovascular events.Methods:1.According to the inclusion criteria,NICE patients(153 in total)consecutively collected in June 2016 to June 2018 in Yongchuan Affiliated Hospital of Chongqing Medical University neurology department,83 of the patients were categorized into the HR-NICE group,and 70 of the patients were in the NHR-NICE group,and another 15 healthy volunteers werechosen as the NC group.We collected demographic characteristics,history data,ABCD2 score,the NIHSS score,mR S score;2.The results of blood routine,liver function,kidney function,cardiac enzyme,total cholesterol,triglycerides,low-density lipoprotein,homocysteine,blood glucose,electrocardiogram,head MRI,MRA or CTA were collected from the enrolled patients.3.Circulating CD34+/VEGFR2+EPCs were detected by flow cytometry.4.ELISA was used to measure the concentrations of serum SDF-1,VEGF,FGF and PDGF-BB.5.Univariate Logistic regression analysis was used to screen out potentially significant variables,and then multivariate Logistic regression analysis was conducted to analyze which variables were independent risk factors for HR-NICE.Results:1.Among the 153 NICE patients,85 were male and 68 were female,with an average age of 59.15 years old,but the HR-NICE group was older and had more patients with hypertension and diabetes(P < 0.01).2.Compared with the NHR-NICE group,patients in the HR-NICE group had higher triglycerides,total cholesterol and LDL(P < 0.05).However,there was no significant difference between the two groups(P >0.05)in blood sampling time,gender,smoking,body mass index and homocysteine.3.Compared with the HR-NICE group,the number of CD34+/VEGFR2+EPCs in the NHR-NICE group was higher(P < 0.01).However,SDF-1α,VEGF and FGF were lower,and the difference between the two groups was significant(P < 0.01).PDGF-BB expression in the two groups was not statistically significant(P=0.139).4.Univariate Logistic regression analysis screened out possible significant variables related to the occurrence of HR-NICE.Factors screened out included age,hypertension,diabetes,triglycerides,total cholesterol,low-density lipoprotein,CD34+/VEGFR2+EPCs,SDF-1α,VEGF,FGF(P < 0.05).5.Multivariate Logistic regression analysis results were age(OR:1.134,95%CI: 1.030-1.249,P=0.011),hypertension(OR: 10.798,95%CI:2.174-53.622,P=0.004),diabetes(OR: 11.630,95%CI: 1.487-90.941,P=0.019),total cholesterol(OR: 2.416,95%CI: CD34+/VEGFR2+EPCs(OR: 0.067,95%CI: 0.023-0.198,P<0.01),SDF-1α(OR:1.007,95%CI:1.003-1.011,P < 0.01),which related to the occurrence of HR-NICE.Conclusions:1.HR-NICE patients were older and had more risk factors for cerebrovascular diseases.2.The decrease of circulating CD34+/VEGFR2+EPCs is one of the reasons for the high risk of HR-NICE.The increased SDF-1α and VEGF can promote the mobilization of EPCs into the blood,and promote the repair of endothelial dysfunction and angiogenesis.3.Age,hypertension,diabetes,total cholesterol,SDF-1α were risk factors for HR-NICE,and CD34+/VEGFR2+ EPCs was protective factor for HR-NICE,all these factors may be predictive factors for HR-NICE.The number of CD34+/VEGFR2+ EPCs and the SDF-1α concentration have important clinical value in predicting the occurrence of HR-NICE.PART Ⅲ EVALUATION OF DIFFERENT SUBTYPES OF ENDOTHELIAL PROGENITOR CELLS FOR NON-DISABLING ISCHEMIC CEREBROVASCULAR EVENTSBackground:EPCs can not be identified morphologically and have different surface markers at different stages of maturation.EPCs are positive for CD133,CD34 and VEGFR2 in the bone marrow stage.After EPCs mobilized into the blood,the expression of CD133 was gradually reduced,while CD34 and VEGFR2 were still expressed.Thereafter,CD34 expression was also gradually reduced during the maturation of EPCs,but VEGFR2 remained positive.Therefore,EPCs is a group of cells with different subtypes.Currently,CD34+/VEGFR2+ cells are considered to be EPCs,and our team has used VEGFR2 to label bone marrow EPCs for flow cytometry detection.Since CD34+/VEGFR2+ cells are used for flow cytometry as a presence of EPCs,which can be predicted the number of circulating EPCs,whether VEFDR2+ cells can also be used for flow cytometry as a presence of EPCs and predicted the number of circulating EPCs.In this study,we investigated another possible clinical measurement of circulating EPCs based on the difference between VEGFR2+EPCs and CD34+/VEGFR2+EPCs.At the same time,to explore whetherVEGFR2+EPCs can also be used as an early risk assessment indicator for HR-NICE.Objective:To explore whether VEGFR2+EPCs can be used as an early risk assessment indicator for risk factors of NICE.Methods:1.According to the inclusion criteria,153 NICE patients who visited the department of neurology of Yongchuan Hospital Affiliated Chongqing Medical University from June 2016 to June 2018 were continuously collected,who were divided into the HR-NICE group and the NHR-NICE group.Another 15 healthy volunteers were selected.Demographic characteristics,medical history,ABCD2 score,NIHSS score and mR S score were collected.2.The results of blood routine,liver and kidney function,cardiac enzyme,total cholesterol,triglycerides,low-density lipoprotein,homocysteine,blood glucose,electrocardiogram,brain MRI,MRA or CTA were collected from the enrolled patients.3.Flow cytometry was used to detect circulating VEGFR2+EPCs and CD34+/VEGFR2+ EPCs.4.According to univariate Logistic regression analysis,possible significant variables were screened out,and then multivariate Logisticregression analysis was conducted to analyze which variables were independent risk factors for NICE.Results:1.The mean values of circulating VEGFR2+EPCs in the HR-NICE group,the NHR-NICE group and the NC group were 1.93%,2.22% and1.44%,respectively,with significant differences among the three groups(P=0.002).However,there was no significant difference between the HR-NICE group and the NHR-NICE group(P=0.39).2.The mean ratio of circulating CD34+/VEGFR2+EPCs accounted for monocytes in the HR-NICE group,the NHR-NICE group and the NC group was 4.09/000,5.75/000 and 3.40/000 respectively,and the difference was significant(P < 0.01).Compared with the NHR-NICE group,the number of circulating CD34+/VEGFR2+EPCs decreased in the HR-NICE group,and the difference was significant(P < 0.01).Compared with the NC group,the circulating CD34+/VEGFR2+EPCs in the HR-NICE group and the NHR-NICE group were higher,and the difference was statistically significant(P < 0.05).3.The difference in circulating VEGFR2+EPCs between the HR-NICE group and the NHR-NICE group was not statistically significant(P >0.05),so the results of VEGFR2+EPCs could not be included in the multivariate regression analysis.Conclusions:1.The number of circulating CD34+/VEGFR2+EPCs decreased,which may lead to NICE convert to HR-NICE.2.Circulating VEGFR2+EPCs decreased in the HR-NICE group,but could not be used as a predictor of whether NICE was converted to HR-NICE.