Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics.
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-1 beta 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-1 beta 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.
Moldovan
F
F
Department of Hormonal Biology, Saint Louis University Hospital, Paris, France.
Benanni
H
H
Fiet
J
J
Cussenot
O
O
Dumas
J
J
Darbord
C
C
Soliman
H R
HR
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport; D002454:Cell Differentiation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000254:growth & development*; D006801:Humans; D010641:Phenotype; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000254:growth & development*
D000952:Antigens, Polyomavirus Transforming; D001120:Arginine; D012964:Sodium