Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated to various pathophysiological conditions such as reperfusion [1], endotoxic shock [2], diabetes [3] and aging [4]. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction might be related to PARP-1 overactivation and ATP depletion. An alternative way in which PARP-1 may influence endothelial cell function could involved transcription regulation [5]. Primary endothelial cells have a limit life span in culture. After 5-6 passages, they tend to de-differentiate and eventually reach senescence which limits their use in studies of endothelial cell function. The aim of this work was to establish an immortalized PARP-1-deficient endothelial cell line. Primary murine heart endothelial cells (MHEC) were isolated from PARP-1 knockout mice [6] (kindly provided by Dr. de Murcia) by collagenase treatment and cell sorting of ICAM-2-positive cells. MHEC were transfected with the pRNS-1 plasmid encoding the SV40 genome (kindly provided by Dr. Ozer) and a clone was selected on the basis of their morphological and phenotypical features. Flow cytometry analysis of this cell line (HYKO6) demonstrated constitutive expression of MHC-Class I, CD105, ICAM-2, and VCAM-1 antigens. Moreover, the HYKO6 cell line was able to up-regulate the expression of E-selectin in response to TNF-a. This cell line has been maintained in culture for many passages without signs of senescence and with preserved characteristics of primary endothelial cells. The HYKO6 cell line will provide a useful tool to study the role played by PARP-1 in endothelial cell dysfunction.References: 1.Szabó C et al: Cir Res, 2002; 90: 100-106 2.Szabó C et al: J Clin Invest, 1997; 100: 723-735 3.García Soriano F et al: Nat Med, 2001; 7: 108-113 4.Pacer P et al: Br J Pharmacol, 2002; 135: 1347-1350 5.Chiarugi A: Trends Pharmacol Sci, 2002; 23: 122-129 6.Menissier de Murcia J et al: Proc Natl Acad Sci USA, 1997; 94: 7303-7307

Keywords: PARP-1, Endothelium, Cell Line