Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

Abstract

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.

Conclusion: 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.

Fig. 1

The construction of pLXSN-SV40 LT and pLPCX-hTERT recombined plasmids SV40 LT and hTERT was subcloned from pMFGSV40 tsLT to EcoRI/BamHI site of pLXSN or pCIneo-hTERT to EcoRI/NotI site of pLPCX respectively

Fig. 2

Positive staining of von Willebrand factor in immortalized endothelial cells (×100)

Fig. 3

The morphology of immortalized endothelial cells under scanning electron microscope (a) The cells were polygonal, round or fusiform (magnification of ×200); (b) (magnification of ×5000) and (c) (magnification of ×6000) There were many apophyses on their surface

Fig. 3

The morphology of immortalized endothelial cells under scanning electron microscope (a) The cells were polygonal, round or fusiform (magnification of ×200); (b) (magnification of ×5000) and (c) (magnification of ×6000) There were many apophyses on their surface

Fig. 3

The morphology of immortalized endothelial cells under scanning electron microscope (a) The cells were polygonal, round or fusiform (magnification of ×200); (b) (magnification of ×5000) and (c) (magnification of ×6000) There were many apophyses on their surface

Fig. 4

Detection of SV40 LT and hTERT with Western blot assay a, c and d were from immortalized endothelial cells, and b, e and f were from normal endothelial cells. The expression of SV40 LT and hTERT could be only detected in immortalized endothelial cells

Fig. 5

The expression of endothelial lipase in immortalized endothelial cells The expression of endothelial lipase was similar with normal HUVECs. After the intervention with TNF-α, the expression of endothelial lipase was not up-regulated significantly. a1~3 were transfected cells; b1~3 were normal cells; t1~3 were transfected cells with the intervention of TNF-α

Fig. 6

The expression of E-selectin in transfected cells exposed to TNF-α When the transfected cells were exposed to TNF-α, the expression of E-selectin in them was up-regulated significantly (38.60±1.83 vs 17.19±0.77, P<0.01, student T-test)