Changes Of Circulating Endothelial Progenitor Cells In Patients With Cyanotic Congenital Heart Diseases

Objective: To investigate alterations of endothelial progenitor cells(EPCs)from peripheral blood in patients with cyanotic congenital heart diseases, and explore the probability of the application of EPCs in the tissue engineered vessels as seed cells.Methods: 20 ml venous blood was obtained from the peripheral blood of patient with Tetralogy of Fallot (TOF) and Ventricular Septal Defect (VSD), each group include 10 cases. The mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolated cells were suspended in Medium 199 supplement with 20 % fetal blood serum (FBS) and bovine pituitary extract (BPE) for culturing, differentiating and proliferating. The expressions of specific antigens on cell surface, such as CD133,KDR,CD34,CD31 and vWF were analyzed by immunocytochemistry on day 10, Cultured cells were further stained with Dil-AcLDL and FITC-UEA-1, transmission electron microscope was used to identify the distinct cell organelles of EPCs: immature mitochondrion and Weibel-Palade body. Flow cytometer was used to quantify the CD133~+/KDR~+ cells, Modified Boyden chamber assay and MTT assay was used to measure the migration and proliferation function of EPCs. The adhesion assay was performed by replating on fibronectin-coated dishes, then adherent cells were counted.Results:1. Surface markers were found to be characteristic for EPCs (vWF~+, CD31~+, CD133~+, CD34~+ and KDR~+).2. Transmission electron microscope identified the distinct cell organelles of EPC: immature mitochondrion and Weibel-Palade body.3. The EPCs Colony number was significantly increased in patients with TOF compared with that of control subjects(14.7±3.1 vs8.2±1.34/×100 field, P＜0.01).4. The number of DiI-AcLDL/FITC-UEA-I double positive cells was significantly increased in patients with TOF compared with that of control subjects (72.2±9.73vs51.2±3.83/×200 field, P＜0.01).5. Flow cytometric measurement of MNCs showed the rate of CD133/KDR double positive cells was (0.66±0.20)% in cyanotic group and (0.18±0.08)% in control group (P＜0.01).6. Migration assay showed that the number of migrating cells in a visual field (200×high-powered-fields) was (141±9) in the cyanotic group and (92±9) in the control group (P＜0.01).7. Cell adhesion assay showed that EPCs in the cyanotic group exhibited a significant increase in the number of adhesive cells (149±12/200×high-powered-fields) at 30 minutes, which was higher than that of the control group (113±7/200×high-powered-fields) (P＜0.01).8. MTT assay revealed a different proliferation potential between the two goups that OD value of cells at 492 nm was (0.338±0.021) in the cyanotic group and (0.266±0.014) in the control group (P＜0.01).Conclusion:1. The number of EPCs is significantly increased in patient with cyanotic congenital heart diseases.2. The migration, adhesion and proliferation potential of EPCs are significantly increased in patient with cyanotic congenital heart diseases.3. The EPCs in patient with cyanotic congenital heart diseases may be used as good seed cells in constructing tissue engineered vessls.