on December 23, 2004
Published online before print December 23, 2004, doi: 10.1161/01.ATV.0000154360.36106.d9
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Submitted on August 4, 2004
Accepted on December 9, 2004
Stable Knock-Down of the Sphingosine 1-Phosphate Receptor S1P1 Influences Multiple Functions of Human Endothelial Cells
Vera Krump-Konvalinkova *;
From the Institute for Prevention of Cardiovascular Diseases (V.K.-K., T.R., W.A., C.V., W.S.), Ludwig Maximilian University, Munich, Germany; the Department of Physiology (S.Y., N.M., G.T.), University of Tennessee Health Science Center, Memphis; the Institute of Medical Microbiology, Immunology and Hygiene (J.M.), Technical University, Munich, Germany; and the Institute of Pathology (C.J.D.), Johannes Gutenberg University, Mainz, Germany.
* To whom correspondence should be addressed. E-mail: vera.krump-konvalinkova{at}klp.med.uni-muenchen.de.
Objectives--Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed.
Methods and Results--All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-
. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing.
Conclusions--Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.
Key words: S1P1 • functional analysis • siRNA • permanent inhibition • endothelial cells
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