The role of poly(ADP-ribose) polymerase (PARP) in the autonomous proliferative response of endothelial cells to hypoxia.
OBJECTIVE
The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation.


METHODS
Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia.


RESULTS
Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor.


CONCLUSIONS
PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.
Abdallah
Yaser
Y
Institute of Physiology, Justus-Liebig-University, D-35392 Giessen, Germany. Yaser.Abdallah@physiologie.med.uni-giessen.de
Gligorievski
Dragan
D
Kasseckert
Sascha A
SA
Dieterich
Lukas
L
Schäfer
Matthias
M
Kuhlmann
Christoph R
CR
Noll
Thomas
T
Sauer
Heinrich
H
Piper
H Michael
HM
Schäfer
Claudia
C
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007150:Immunohistochemistry; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010616:Phenanthrenes / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine
D002070:Butadienes; C434926:N-(oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride; D009570:Nitriles; D016376:Oligonucleotides, Antisense; D010616:Phenanthrenes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D017382:Reactive Oxygen Species; C113580:U 0126; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C086986:CYBA protein, human; D011065:Poly(ADP-ribose) Polymerases; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium