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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:546.)
© 2005 American Heart Association, Inc.

Vascular Biology

Stable Knock-Down of the Sphingosine 1-Phosphate Receptor S1P₁ Influences Multiple Functions of Human Endothelial Cells

Vera Krump-Konvalinkova; Satoshi Yasuda; Tina Rubic; Natalia Makarova; Jörg Mages; Wolfgang Erl; Claudia Vosseler; C. James Kirkpatrick; Gabor Tigyi; Wolfgang Siess

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.

Correspondence to Dr Vera Krump-Konvalinkova, Institute for Prevention of Cardiovascular Diseases, Universität München, Pettenkoferstr 9, D 80336 München, Germany. 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 S1P_1-5 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.

Stable silencing by RNA interference of S1P₁ expression in human endothelial cell lines demonstrates the involvement of S1P₁ in key functions of human endothelial cells and identifies new S1P₁-dependent gene targets.

Key Words: S1P₁ • functional analysis • siRNA • permanent inhibition • endothelial cells

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