The Number And Function Of Endothelial Progenitor Cell Population In Coronary Artery Spasm

AbstractBackground:Coronary artery spasm(CAS)plays an essential role in the pathogenesis of vasospastic angina,myocardial infarction,sudden death,and other ischemic heart diseases.It has been identified that endothelial dysfunction presents a main pathophysiological cause for coronary spasm.Precious studies showed that the vascular endothelium has been considered as a multifunctional organ,which exhibits complex metabolic capabilities.Thus,it is important for normal vascular function to maintain the integrity of the vascular endothelium and a well-balanced release of vasoactive substances.Increasing evidences suggest that the injured endothelial monolayer may be regenerated by endothelial progenitor cells(EPCs),which accelerates re-endothelialisation.Reduced levels and function of circulating EPCs significantly afford to endothelial dysfunction.EPCs have been classified as early EPCs and late outgrowth endothelial cells(OECs).Nevertheless,no study has reported the function of EPCs in the development of CAS.1.Objective:To investigat the role of the different EPCs populations in human coronary artery spasm.2.Methods:2.1.Patients and controlsIn this study,we recruited population between June 2016 and December 2016.All consecutive patients admitted to the Second Affiliated Hospital of Nantong University were divided into coronary artery spasm group(30 patients)fulfilled the criteria according to the Japanese Circulation Society Guidelines for Diagnosis and Treatment of Patients with Vasospastic Angina(Coronary Spastic Angina)(JCS 2013),and the finding of<50%stenosis in major coronary artery on coronary angiography,coronary artery disease group(22 patients),least one lesion with>70%stenosis in major coronary artery on coronary angiography,and control group(20 patients),who had experienced atypical chest pain,without the finding of<50%stenosis in major coronary artery on coronary angiography and proved negative on acetylcholine provocation test.2.2 EPC isolation,cultivation and quantificationAfter placement of an arterial sheath,50 mL of whole blood obtained for EPCs isolation.Mononuclear cells(MNCs)were isolated by density gradient separation.MNCs were planted into six-well tissue culture plates precoated with human fibronectin and cultured in endothelial cell growth medium-2MV.EPCs were quantificatd based on cell culture,immunofluorescence and flow cytometry methods.2.3 EPCs number detectionThe number EPCs and outgrowth endothelial cells(OECs)were determined in peripheral blood samples by flow cytometry methods.After 7 and 21 days of culture,EPCs colonies were calculated by scanning culture plates.2.4 EPCs function assayThe function of early EPCs was determined by measuring proliferation assay,migration assay,tube formation assay and NO production.2.5 Growth factor detectionThe cytokines VEGF165(vascular endothelial growth factor)and SDF-1(stromal cell-derived factor-1)were analyzed by ELISA(enzyme-linked immunosorbent assay).2.6 Western blot and RT-PCRAfter 7 and 21 days of culture,the protein expressions of eNOS,p-eNOS were examined by Westin blot analysis,and RT-PCR was used to detect the level of eNOS mRNA.2.7 Flow-mediated vasodilationFlow-mediated vasodilation(FMD)was evaluated using Sonos 5500 to scan the brachial artery.FMD was calculated as the maximal post occlusion diameter relative to the averaged preocclusion diameters.2.8 Statistical analysisAll data were showed as mean ± SD for numeric variables and as number(percentage)for categorical variables.p<0.05 was considered significant.Differences in baseline characteristics of underlying diseases,and treatments were compared using X2 test.The correlation among independent variables was analyzed by performing simple linear regression analyses.3.Results3.1 Patients' baseline characteristics.In this study,seventy-two patients were recruited for the study.No significant differences were found among the CAS,CAD,and control group in baseline characteristics considering age,sex,body mass index,smoking,systolic blood pressure,diastolic blood pressure,diabetes,and serum levels of total cholesterol,triglyceride,high-density lipoprotein,low-density lipoprotein and creatinine.No significant differences were found in drugs administrated,including aspirin,angiotensin-converting enzyme inhibitors(ACEI),angiotensin II receptor blockers and statins(ARB).3.2.Characterization of EPCs.The initially planted cells were round.Up to the seventh day,the cells began to be clustersand these cells were called early EPCs.Then,we found that OECs appeared as clusters on 21 day after plating,and showed cobblestone appearance.Both types of EPCs took-up DiI-acLDL and showed lectin binding affinity.Finally,we determined the CD34?CD 133 and KDR in EPCs by using flow cytometry.Compared with the EPCs on 7 day,we observed that CD 133 was significantly downregulated about 50%and KDR was upregulated more than 80%in EPCs on the 21 day.3.3.The number and function of EPCs in CAS,CAD and control groups.Compared with controls,patients with coronary spasm group(CAS)were found to have significantly decreased in CD34+/CD45-,OECs colonies,OECs proliferation,and OECs tubulogenesis.However,no differences were noted in early EPC colonies,CD34+/KDR+,CD34+/CD45+,early EPC migration and OEC migration.On the other hand,we found that patients with coronary artery disease(CAD)were found to have significantly decreased in CD34+/KDR+,CD34+/CD45+,CD34+/CD45-,early EPCs colonies,OECs colonies,early EPCs proliferation,OECs proliferation,early EPCs migration,OECs migration,and OECs tubulogenesis compared with controls.3.4.The NO and eNOS in the CAS,CAD and control groups.Patients with CAS and CAD were found to exhibit obviously downregulated NO production,eNOS,and the phosphorylation(Ser1177)of eNOS in OECs compared with controls.Similarily,patients with CAD had significantly decreased NO production,eNOS,and the phosphorylation(Ser 1177)of eNOS in early EPCs compared with the control group.However,there was no difference of the variables in early EPCs between CAS and controls.3.5 Relationship between FMD and EPCs levels and functionsThe results showed that patients with CAS and CAD had significantly decreased FMD compared with the control group,and there were no difference between CAS and CAD.Further analysis demonstrated that FMD was significantly correlated with OECs NO production,OECs eNOS phosphorylation(Ser 1177),OECs colonies,and OECs proliferation.3.6 Relationship between growth factors and EPC number and functionsNo significant differences were showed in plasma VEGF165 and SDF-1 concentration in the three groups,and no significant correlation was found between plasma VEGF165 and(or SDF-1)concentration and EPC number and function.4.Conclusion? The number and function of OECs were found abnormality in the spasm group.? No significant correlation was found between plasma VEGF165(or SDF-1)concentration and EPC number and function in the spasm group.? Endothelial function is abnormal in patients with CAS.? The number and function of OECs play a central role in the endothelial function of patients with CAS.