Changes Of Circulating EPCs Numbers And Function In Children With Cyanotic Congenital Heart Defects: The Study Of Mechanism

PartⅠCorrelation of the Serum Levels of VEGF and SDF-1 and numbers and function of circulating EPCs in Children With cyanotic congenital heart defectsObjective:To examine numbers and function of circulating EPCs in cyanosis congenital heart diseases patients and discuss their correlation with serum levels of VEGF and SDF-1.Methods:The study group comprised 20 patients(12 with TOF,6 with DORV and 2 with TGA).The control group consisted of 20 acyanotic congenital heart diseases(15 with VSD and 5 with ASD),age and sex matched.The numbers and function of Circulating EPCs was assessed on the basis of specialist procedures.Serum levels of VEGF and SDF-1 were evaluated by ELISA.And linear regression was used,and analysed Correlation of Serum Levels of VEGF and SDF-1and numbers and function of circulating EPCs.Results:We found that the changes of numbers and function of EPCs is raised in 20 patients(12 with TOF and 6 with DORV and 2 with TGA),and We also observed that higher serum levels expression of VEGF and SDF-1 were demonstrated in cyanotic patients in comparison to the control group,namely VEGF(ng/L):201.42±44.74 vs 113.56±35.62,P＜0.05;SDF(ng/L):3.4535±1.0732 vs 1.05368±0.98692,P＜0.05;The number of EPCs was significantly increased in patients with cyanotic patients compared with that of control subjects(5915±1016 vs 3118±717 EPCs×200 field;P＜0.05).In addition,the functional activity of EPCs such as proliferation,migration and adhesive capacity was also augmented in patients with cyanotic patients.1.proliferation capacity:0.3377±0.0205 vs 0.2657±0.0142,OD 490nm,P＜0.01;2.migration capacity:146.6±9.24 vs 91.84±8.58,×200 field,P＜0.01;3.adhesive capacity:149±11.58 vs 112.6±7.02,×200 field,P＜0.01.Also higher the numbers and function of EPCs and higher Serum Levels of VEGF and SDF-1 in cyanotic patients is consistent,and correlation coefficient is 0.675,0.835,0.724 respectively.Conclusion:1.There is a number of circulating EPCs in the peripheral blood of Children With complicated cyanotic congenital heart defects,and the numbers and function of circulating EPCs was significantly increased in patients with cyanotic patients,so the circulating EPCs may be used one of the source of autologous seed cells.2.The Serum Levels of VEGF and SDF-1 in cyanotic patients was significantly increased,and may promote the change of numbers and function of circulating EPCs.3.The VEGF and SDF-1 maybe play an important role in the Pathology and physiology in patients with cyanosis congenital heart diseases. PartⅡThe correlation analysis between the expression of HIF-1a in myocardial tissue and the Serum Levels of VEGF and SDF-1 in cyanotic patientsObjective:To detect the expression of HIF-1a in myocardial tissue, and the serum levels of VEGF and SDF-1 in cyanotic patients and discuss the relation between the HIF -1a and VEGF,SDF-1.Methods:Immunohistochemistry,RT- PCR and Western blot were used to investigate the expression of HIF- 1a in RV myocardium obtained during surgical repair of congenital heart defects from 40 patients:20 cyanotic and 20 acyanotic.And the Serum levels of VEGF and SDF-1 were evaluated by ELISA.And linear regression was used,and analysed the correlation of HIF-1a and VEGF,SDF-1.Results:The mRNA and protein of HIF- 1a could be detected in RV myocardium obtained during surgical repair of cyanotic patients,and the expression of HIF- 1a could not be detected in control group.Higher serum levels expression of VEGF,SDF-1 were demonstrated in cyanotic patients in comparison to the control group.Namely VEGF(ng/L): 201.42±44.74 vs 113.56±35.62,P＜0.05;SDF(ng/L):3.4535±1.0732 vs 1.05368±0.98692,P＜0.05;the mRNA and protein expression of HIF-1a were higher in cyanotic patients in comparison to the control group, and higher the mRNA and protein expression of HIF-1a and higher the Serum Levels of VEGF and SDF-1 in cyanotic patients is consistent,and correlation coefficient is 0.626.Conclusions:1.HIF-1a in chronic myocardial hypoxia tissue exists over-expression.2.HIF-1a plays an important role under hypoxia,and regulating the expression of VEGF and SDF-1.3.The over-expressions of HIF-1a and the higher Serum Levels of VEGF and SDF-1 have closely relation,and maybe regulate the change of Pathology and physiology in cyanotic patients. PartⅢEffect of HIF-1a in the proliferation and migration of Endothelial Progenitor Cells in ex vivoObjective:To investigate the effect of over-expressing HIF-1a in the proliferation capability and migration capability of Endothelial Progenitor Cells,and discuss the possible mechanism ex vivo.Methods:Peripheral blood mononuclear cells obtained from the patients with acyanotic congenital heart diseases were cultured in EPCs medium,and harvested 3d later,We transfected over-expressed HIF-1a to EPCs and Immunofluorescence technique was carried out to measure the efficienty of HIF-1a transfected EPCs,and measured HIF-1a and VEGF level with RT-PCR after 6h,12h,24h,48h,72h of transfection,the numbers and function of EPCs was assessed on the basis of specialist procedures,and Conditioned medium was assayed for VEGF by ELISA.Results:HIF-1a were transfected in EPCs DNA in normoxia,and EPCs could be induced by over-expressing HIF-1a,and the HIF-1a controlled target gene VEGF was induced after HIF-1a transfection, Immunofluorescence technique showed that human EPCs expressed HIF-1a protein after transfection,HIF-1a and VEGF was enhanced by over-expressed HIF-1a.A time kinetic VEGF was measured by ElISA, which was markly enhanced by HIF-1a(P＜0.05),and the functional activity of EPCs of HIF-1a cDNA such as proliferation,migration capacity was also augmented in HIF-1a transfected EPCs,. Conclusion:1.HIF-1a maybe plays an important role in raising numbers and function of EPCs under hypoxia.2.transcription factor HIF-1a is a key determinant of oxygen-dependent gene regulation,and poromate the expression of VEGF.