Experimental study of recombinant eukaryotic expression vector of human eNOS in ECV304.
OBJECTIVE
Gene transfer with recombinant non-viral vectors encoding vasodilator proteins, such as endothelial nitric oxide synthase (eNOS), maybe a preferential choice in gene therapy of artery restenosis following angioplasty, stent or anastomosis. However, the transfection rate of a non-viral vector, the harmful effects of eNOS transfection on endothelial cells (EC) and the control release of nitric oxide (NO) have been controversial. We designed the eukaryotic expression vector pcDNA3.1-eNOS to study the regulated expression of eNOS (in the presence of various chemical agents) and to evaluate the exogenous NO effect on EC proliferation in vitro.


METHODS
The full-length human eNOS cDNA was inserted into the EcoRI cloning site of the pcDNA3.1 expression plasmid and the eNOS direction was tested by restriction enzyme digestion with XhoI to construct recombinant pcDNA3.1-eNOS. After co-transfection of pcDNA3.1-eNOS with pcDNA3.0-EGFP mediated by cationic liposomes into Human umbilical vein endothelial cells (ECV304), the transfection rate and the effect on ECV304 proliferation were calculated by fluorescence microscopy and flowcytometry. eNOS mRNA and protein were detected by reverse transcription-PCR (RT-PCR) and immunofluorescence, respectively. The eNOS activity, NO release and changes of the relevant cells growth curve were assessed after treating the transfected cells with four independent factors including ie Ca2+, L-arginine (L-Arg), Ethylene Diamine Tetraacetic Acid (EDTA) and N-nitro-L-arginine methylester (L-NAME). In addition, we examined the non-transfected cells status by isolated sodium nitroprusside (SNP) treatment.


RESULTS
eNOS cDNA was inserted into pcDNA3.1 in the proper direction. RT-PCR analysis showed that pcDNA3.1-eNOS transfected cells could express eNOS mRNA. The rate of eNOS transfection was 39.6 +/- 3.4%. Immunofluorescence staining displayed that subcellular localisation of eNOS was most prominent in plasma membrane and perinuclear regions of the cell. The eNOS activity of eNOS transfected cells had not increased significantly, whereas, in the presence of Ca2+, L-Arg, EDTA, and L-NAME, the eNOS activity was 96.98 +/- 13.47, 32.57 +/- 6.39, 11.63 +/- 3.02, 15.56 L 7.34 U/ml respectively and the NO level was 55.34 +/- 11.19, 9.43 +/- 4.51, 2.63 +/- 1.41, 3.73 +/- 1.65 mmol/L, respectively. Meanwhile, the growth curves of EC shifted. SNP also had obvious growth-inhibiting effects on the cells. Together, the ECV304 growth curve went downward in a NO concentration-dependent manner.


CONCLUSIONS
Eukaryotic expression vector pcDNA3.1-eNOS was constructed successfully with the ability to express human eNOS mRNA and protein in EC effectively. The activity of eNOS in EC could be regulated by certain exogenous factors. Ca2+ was an important factor promoting NO release and excess NO had a cytotoxic effect on EC in vitro. Controlled release of NO in vivo and polygenic measurements might be considered in more clinical gene therapy studies.
Qiao
Tong
T
Department of Vascular Surgery, Affiliated Drum Tower Hospital of Medical school, Nanjing University, China. qiaotongmail@yahoo.com.cn
Liu
Chang-jian
CJ
Ran
Feng
F
Han
Li
L
Zhang
Le
L
Li
Lei
L
D002460:Cell Line; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D015870:Gene Expression; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014471:Umbilical Veins
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III