Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics |
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Authors: | Florina Moldovan Houda Benanni Jean Fiet Olivier Cussenot Jacques Dumas Christian Darbord Hany R Soliman |
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Institution: | (1) Department of Hormonal Biology, Saint Louis University Hospital, 1, C. Vellefaux Avenue, 75010 Paris, France;(2) Department of Urology, Saint Louis University Hospital, 1, C. Vellefaux Avenue, 75010 Paris, France;(3) Biotechnology Department Roussel-UCLAF Laboratories Paris V University, France;(4) Department of Microbiology Faculty of Biological Sciences, Paris V University, France |
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Abstract: | Summary The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical
vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan,
was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator
(t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE).
In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted
interleukins (IL-1β and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive
peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the
immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted
the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which
cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability
to express the functional specific amino acid Na+-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF,
nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without
alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation,
and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to
study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1β and IL-6 secretions, and gene
regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary
endothelial cells both from human and animal origin. |
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Keywords: | immortalization endothelial cells endothelin interleukins arginine SV40 |
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