| Research background:The declined function of endothelial progenitor cells (EPCs) in patients with coronary heart disease is an important factor that affects the endothelial repair and leads to restenosis after stent implantation. We discovered recently that the increased level of asymmetric dimethylarginine (ADMA), an inhibitor of exogenous nitric oxide synthase (NOS) was closely associated with the declined function of EPCs in patients with coronary heart disease. The dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme in the ADMA metabolism in vivo, which declines functionally and leads to increase of ADMA in atherosclerosis-linked diseases. Both of them have an influence on the functional status of cardiovascular system in increase and decrease states. Our study was planned to reconstruct EPCs with DDAH2over-expression, and observe the effect of DDAH2over-expression on EPCs function; further observe the influence of transplanted hEPCs with DDAH2over-expression on vascular reendothelialization and intimal hyperplasia in nude rat model with balloon injuries; and discuss the relationship between DDAH/ADMA system and EPCs functional changes. Our study will help reveal the mechanism of EPCs functional injuries and provide a new thinking for preventive control of restenosis.Methods:The experiment including three procedures:(1) EPCs separation and identification:mononuclear cells of human umbilical cord blood were separated using density gradient centrifugation technique, cultured for10d, and identified by double staining approach using acetylated low density lipoprotein (Dil-acLDL) and Ulex lectin1(FITC-UEA-1). The markers CD133, CD34and vWF were detected using flow cytometry to further confirm EPCs.(2) The effect of DDAH2over-expression on EPCs function:the lentiviral vector for DDAH over-expression was reconstructed under assistance of Shanghai Jikai Gene Chemical Technology Co., Ltd.; the transfection efficiency was determined by using fluorescent and RT-PCR technique; the adhesion function of EPCs on human fibronectin and endothelial cell were tested with cell counting method. The cell migration of EPCS was measured by using a modified Boyden chamber.(3) The effect of DDAH2over-expression EPCs on reendothelialization process and intimal hyperplasia after balloon injuries in nude rats:reendothelialization was tested by using Evans blue staining method; and intimal hyperplasia was determined by using HE staining method.Results:(1) EPCs with DDAH2over-expression improved the adhesion to the human fibronectin (FN) significantly, P<0.01compared to the control groups while the EPCs transfected with non-targeted gene (non T-EPCs) improved the adhesion to the human fibronectin insignificantly; EPCs with DDAH2over-expression increased the adhesion to the TNF-a-induced endothelial cells significantly, P<0.01compared to the control group, while EPCs transfected with non-target gene (non T-EPCs) didn’t show the similar effects.(2) DDAH2over-expression improved the migration capability of SDF-1-induced EPCs significantly, P<0.01compared to the control group, while EPCs transfected with non-target gene (non T-EPCs) didn’t show the similar effects.(3) Transplanted EPCs with DDAH2over-expression after balloon injuries could increased the rate of reendothelialization significantly, P<0.01compared to the control group (locally injected with PBS solution).(4) Transplanted EPCs with DDAH2over-express could inhibit carotid intimal hyperplasia in nude rats significantly,P<0.01compared to the control group (locally injected with PBS solution).Conclusion:(1) Our study firstly confirmed that EPCS with DDAH2over-expression increased the adhesion and migration significantly;(2) Our study reconstructed the nude rat balloon injuries model successfully, firstly discovered that the transplanted EPCs with DDAH2 over-expression could inhibit carotid intimal hyperplasia and accelerate the reendothelialization process after carotid balloon injuries in nude rats. |
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