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-α. 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.