Endothelial Sphingosine-1-Phosphate Receptor 4 Regulates Blood-Brain Barrier Permeability and Promotes a Homeostatic Endothelial Phenotype

The precise regulation of blood-brain barrier (BBB) permeability for immune cells and blood-borne substances is essential to maintain brain homeostasis. Sphingosine-1-phosphate (S1P), a lipid signaling molecule enriched in plasma, is known to affect BBB permeability. Previous studies focused on endothelial S1P receptors 1 and 2, reporting a barrier-protective effect of S1P1 and a barrier-disruptive effect of S1P2. Here, we present novel data characterizing the expression, localization, and function of the S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). Hitherto, the receptor was deemed to be exclusively immune cell associated. We detected a robust expression of S1P4 in homeostatic murine BMECs (MBMECs), bovine BMECs (BBMECs), and porcine BMECs (PBMECs) and pinpointed its localization to abluminal endothelial membranes via immunoblotting of fractionated brain endothelial membrane fragments. Apical S1P treatment of BMECs tightened the endothelial barrier in vitro , whereas basolateral S1P treatment led to an increased permeability that correlated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels (MBMVs) after stroke, a neurologic disease associated with BBB impairment. RNA sequencing and qPCR analysis of BMECs suggested the involvement of S1P4 in endothelial homeostasis and barrier function. Using S1P4 knock-out (KO) mice and S1P4 siRNA as well as pharmacological agonists and antagonists of S1P4 both in vitro and in vivo , we demonstrate an overall barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic potential in CNS diseases associated with BBB dysfunction. SIGNIFICANCE STATEMENT Many neurologic diseases including multiple sclerosis and stroke are associated with blood-brain barrier (BBB) impairment and disturbed brain homeostasis. Sphingosine-1-phosphate receptors (S1PRs) are potent regulators of endothelial permeability and pharmacological S1PR modulators are already in clinical use. However, the precise role of S1P for BBB permeability regulation and the function of receptors other than S1P1 and S1P2 therein are still unclear. Our study shows both barrier-disruptive and barrier-protective effects of S1P at the BBB that depend on receptor polarization. We demonstrate the expression and novel barrier-protective function of S1P4 in brain endothelial cells and pinpoint its localization to abluminal membranes. Our work may contribute to the development of novel specific S1PR modulators for the treatment of neurologic diseases associated with BBB impairment.