Function Differences And Molecular Mechanism Of Endothelial Progenitor Cells From Patients With Acutemyocardial Infarction And Heart Failure

Background:Heart failure(HF)is a disease of progressive deterioration,which is the most important death factor in recent years.HF has the high prevalence and mortality,so it has become a global epidemic in the 21st century,seriously endangering human health and increasingly impacting on the health care system.The main causes include population aging,myocardial infarction patients have improved survival rates but with left ventricular remodeling,hypertension prevalence and morbidity increased but often poor blood pressure control.In recent years,acute myocardial infarction developing into heart failure,which accounts for a major position in all heart failure diseases.In the global heart failure studies,myocardial infarction(MI)patients account for about half.Acute myocardial infarction(AMI)can cause myocardial necrosis and apoptosis,which is a precursor of heart failure.In the event of heart failure,the heart will enter a process of failure and deterioration,abnormal ventricular remodeling cannot be reversed.Therefore,the prevention and treatment of heart failure should be advanced,the effective treatment and control of primary disease for example hypertension,type 2diabetes,obesity and ventricular remodeling after AMI to prevent or delay the occurrence of HF and prolong life is important.Hypertension,type 2 diabetes have been effectively treated with the developing of medicine and the improving of public health awareness,but prevention of myocardial remodeling after MI and HF are still widespread,which have not been effectively treated.Recent studies have found that stem and progenitor cells could postpone the progress of cardiovascular disease,so that more scholars are beginning to focus on the treatment of cardiovascular disease by regenerative medicine.Especially endothelial progenitor cells(EPCs)become the potential therapeutic agents for improving compensatory mechanisms in the heart and vascular system.Previous EPCs on the treatment of cardiovascular disease results show that autologous EPCs transplantation could improve left ventricular myocardial function in the myocardial infarction model.The recent research of Arshed also confirmed that through intracoronary autologous CD34~+cells implantation can reduce the incidence of adverse cardiac events,and improve heart function in acute myocardial infarction.Obviously,EPCs is a promising candidate in cardiovascular repair.The mobilization of EPCs from stem or progenitor cells niches into the peripheral circulation to participate in postnatal vasculogensis in the injury site.We explored the migration,angiogenesis,and related factors of EPCs in patients with AMI and HF.For determining where we should intervene to enhance the ability of EPCs to repair blood vessels,furthermore delay or prevent the development of heart failure.The cells with the feature of EPCs were isolated and cultured from human arterial blood by in vitro amplification method successfully.We also established a complete process of EPCs identification and amplification.To understand more about the functional changes of EPCs in the development of acute myocardial infarction to heart failure.In this study,EPCs were isolated from healthy people(n=4),AMI patients(n=3,two patients with AMI within 24 hours,one patient with AMI for 6 days)and HF patients(n=3).The EPCs migration,angiogenesis ability and related factors were observed in different patients after in vitro expansion.Objective:To establish a stable platform for the isolation,identification and expansion of EPCs from human arterial blood in vitro,we obtained the arterial blood from healthy people,AMI within 24 hours and HF,and compared differences between functional and molecular mechanism of EPCs from HF and AMI.Methods:1.EPCs isolation:Arterial blood was collected from healthy people,patients with AMI within 24hours and chronic HF.Mononuclear cells were isolated by density gradient centrifugation and cultured by EGM-2 medium in vitro.2.Characterization of EPCs:The morphological structure of EPCs was observed under optical microscope.FACS analyzed the expression of CD133,CD34 and CD31.Confocal immunofluorescence microscopy detected the expression of vWF and Dil-ac-LDL and FITC-UEA-Ⅰ.Matrigel assay identified angiogenesis of EPCs.3.Wound-healing cell migration assays:EPCs of patients with AMI,HF and healthy people were performed the wound healing and calculate the migration rate of various EPCs.4.In vitro migration assay(Transwell):Comparison of migratory capacity of EPCs in patients with AMI and HF.5.Tube formation:The tube forming capacity in vitro was measured in a Matrigel tube-formation assay,which observed at different time points.6.Quantitative RT-PCR:SDF-1αand its receptor CXCR4,eNOS and VEGFA was determined by threen-step RT-PCR.7.Nitric oxide detect(NO):The expression of NO in plasma was detected by NO test kit.Results:1.EPCs were successfully isolated from human arterial blood.Under the optical microscope,cells can form long spindle-shaped,referred to as early endothelial progenitor cells and pebble-like,referred to as late EPCs.EPCs were isolated from healthy people and patients with HF can form microvascular structure in matrix.2.Flow cytometry to identify EPCs surface antigen result showed that it not only expressed EPCs major surface antigen marker CD34,but also expressed endothelial-related surface antigen marker CD31,but CD133,surface antigen of stem/progenitor cell almost did not express.3.Immunofluorescence identification of EPCs marker showed that the expression of vWF was observed by confocal microscopy,which indicating that progenitor cells gradually showed the characteristics of endothelial cells.EPCs were stained with Dil-ac-LDL showed red fluorescence under confocal microscopy Fluorescence,and FITC-UEA-I showed green fluorescence.Differentiated EPCs were stained by Dil-ac-LDL and FITC-UEA-I to show yellow fluorescence under confocal microscopy.4.Wound healing test results showed that EPCs from AMI wound healing faster than EPCs from HF,the EPCs were treated with serum or no.5.Cell migration experiments showed that migration rate of the EPCs from patients with AMI is higher than from patients with HF.6.Tube formation test results showed that EPCs from patients with AMI had the capacity to form tube structure,even if it was treated by its own serum;EPCs from patients with HF can form vascular structure in vitro;EPCs from patients with AMI could form tube structure,if it was treated by 30%serum of patients with HF.7.Quantitative RT-PCR showed that the expression of SDF-1 and eNOS of EPCs from patients with AMI and HF were higher than healthy people.The expression of CXCR4 in patients with AMI was higher than patients with HF,but they were higher than healthy people.The expression of VEGFA in patients with AMI was lower than in healthy people and patients with HF.After EPCs were treated by themselves serum,the expression of SDF-1,CXCR4 and e NOS of EPCs from patients with AMI was higher than in patients with HF and healthy.The expression of VEGFA in patients with AMI and HF was not statistical differences.8.Detection of NO showed NO expression of patients with AMI was higher than patients with HF.Conclusion:EPCs can be successfully isolated from human arterial blood.The migration ability of EPCs from patients with AMI within 24 hours is stronger than patients with HF.The angiogenesis ability of EPCs from patients with HF is stronger than patients with AMI within 24 hours.There are factors that promote the tube formation of EPCs in serum of patients with HF.With the development of AMI,the migration ability of the EPCs from patients with AMI is weakened and the angiogenesis ability is enhanced.