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. 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.

免疫细胞与血液源性物质的血脑屏障（blood-brain-barrier, BBB）通透性精准调控，是维持脑内稳态的必要条件。1-磷酸鞘氨醇（sphingosine-1-phosphate, S1P）作为一种富集于血浆的脂质信号分子，已知可影响BBB通透性。此前的研究多聚焦于内皮细胞的1-磷酸鞘氨醇受体1与2（S1P1、S1P2），并报道S1P1具有屏障保护作用，而S1P2则会产生屏障破坏效应。本研究首次对此前仅被认为与免疫细胞相关的1-磷酸鞘氨醇受体4（S1P4）在原代脑微血管内皮细胞（brain microvascular endothelial cells, BMECs）上的表达、定位及功能特征进行了系统表征。我们在稳态脑微血管内皮细胞中检测到S1P4的高表达，并通过电子显微镜明确其定位于内皮细胞基侧膜。对脑微血管内皮细胞进行基底侧S1P给药后，体外实验显示其屏障通透性升高，该效应与S1P4的表达下调密切相关。同样地，在脑卒中——一种伴随BBB功能受损的神经系统疾病——小鼠的脑微血管中，也可检测到S1P4的表达下调。对脑微血管内皮细胞的RNA测序分析结果表明，S1P4参与内皮细胞稳态、顶基极性及屏障功能的调控。通过在体内外使用小干扰RNA（siRNA）、S1P4的药理学激动剂与拮抗剂，我们证实了S1P4具备屏障保护功能。因此，我们提出S1P4是调控BBB通透性的全新靶点，并建议将其靶向干预应用于伴随BBB功能障碍的中枢神经系统（CNS）疾病。整体实验设计：对小鼠原代脑微血管内皮细胞（MBMECs）分别采用具有屏障开放功能的试剂（50nM S1P 基底侧给药）、屏障收紧功能的试剂（50nM S1P 腔侧给药，或500nM S1P4激动剂腔侧与基底侧联合给药）进行处理，同时设置相应溶剂对照，于处理24小时后对MBMECs开展RNA测序分析。实验设置4组生物学重复，每组使用12只C57/B6小鼠来源的MBMECs。同时纳入经溶剂对照培养基处理的全身性S1P4基因敲除（KO）MBMECs（4组重复，每组12只小鼠）作为对照进行比较。