Transcriptional regulation in primary mouse brain microvascular endothelial cells following apical or basolateral pharmacological treatment with S1P or S1P4 agonist in C57B/6J wildtype versus global S1P4 KO mice

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 focussed 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 hitherto exclusively immunce cell-associated S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). We detected a robust expression of S1P4 in homeostatic BMECs and pinpointed its localization to abluminal endothelial membranes by electron microscopy. Basolateral S1P treatment of BMECs led to increased permeability in vitro associated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels after stroke, a neurological disease associated with BBB impairment. RNA sequencing analysis of BMECs suggested involvement of S1P4 in endothelial homeostasis, apicobasal polarity and barrier function. Using siRNA, pharmacological agonists and antagonists of S1P4 both in vitro and in vivo, we demonstrate a barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic targeting in CNS diseases associated with BBB dysfunction. Overall design: RNA sequencing analysis of MBMECs was performed 24h after pharmacological treatment with functionally barrier-opening (50nM S1P basal) or barrier-tightening (50nM S1P apical or 500nM S1P4 agonist apical and basal) agents or the respective vehicle control. n = 4 sets (replicates) of C57/B6 MBMECs, 12 mice/n. Global S1P4 KO MBMECs (4 replicates, 12mice/n) treated with vehicle control medium were included for comparison.