Establishment of an immortalized PARP-1−/− murine endothelial cell line: A new tool to study PARP-1 mediated endothelial cell dysfunction
✍ Scribed by Ana Carrillo; Yolanda Monreal; Pablo Ramirez; Eduardo Suarez; Pascual Parrilla; Josiane Menissier-de Murcia; Gilbert de Murcia; Luis Alvarez-Vallina; José Yélamos
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 430 KB
- Volume
- 94
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Abstract
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‐α treatment, HYKO6 cells exhibited increased expression of E‐selectin and VCAM‐1. Likewise, NF‐κB‐dependent transcriptional activation was increased in the HYKO6 cell line in response to TNF‐α 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. © 2005 Wiley‐Liss, Inc.