Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen.
OBJECTIVE
To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen.


METHODS
Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.


RESULTS
The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-alpha (tumor necrosis factor-alpha), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found.


CONCLUSIONS
Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.
Department of Cardiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Chang
C
Bian
Kui
K
Zhao
Guo-xin
GX
Tong
Yong-liang
YL
Zhu
Peng
P
Chen
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D048429:Cell Size; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection / Q000379:methods; D014471:Umbilical Veins / Q000166:cytology / Q000502:physiology
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019098:Telomerase