Establishment of an immortalized PARP-1-/- murine endothelial cell line: a new tool to study PARP-1 mediated endothelial cell dysfunction.
Poly(ADP-ribose) polymerase-1 (PARP-1) plays a critical role in endothelial cell dysfunction associated with various pathophysiological conditions. To elucidate PARP-1 pathways involved in endothelial cell dysfunction, it is essential to establish "in vitro" experimental models using isolated endothelial cells. So far, two approaches have been used: primary endothelial cells from PARP-1-/- mice which have a limited life-span, being a major handicap if large quantities of cells are required; and pharmacological inhibition of PARP in PARP-1+/+ endothelial cell lines, which is not specific for PARP-1 and would have biological effects different that genetic inhibition. To overcome these limitations, we have established an immortalized PARP-1-/- endothelial cell line (HYKO6) by transfection of primary cells with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. The HYKO6 cell line exhibited endothelial characteristics, such as constitutive expression of CD105, CD31, ICAM-2, VCAM-1, and von Willebrand factor and formation of capillary-like structures (CLS) on Matrigel surface. However, expression of ICAM-1 antigen is lost in the HYKO6 cells. After TNF-alpha treatment, HYKO6 cells exhibited increased expression of E-selectin and VCAM-1. Likewise, NF-kappaB-dependent transcriptional activation was increased in the HYKO6 cell line in response to TNF-alpha at a level similar to that found for primary PARP-1-/- cells. This cell line should provide, for the first time, a valuable tool to study PARP-1 pathways in endothelial cell dysfunction.
Carrillo
Ana
A
Transplant Unit, University Hospital Virgen de la Arrixaca, Murcia, Spain.
Monreal
Yolanda
Y
Ramirez
Pablo
P
Suarez
Eduardo
E
Parrilla
Pascual
P
Menissier-de Murcia
Josiane
J
de Murcia
Gilbert
G
Alvarez-Vallina
Luis
L
Yélamos
José
J
D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology; D051379:Mice; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017931:DNA Primers; D014409:Tumor Necrosis Factor-alpha; D011065:Poly(ADP-ribose) Polymerases