Rationale: Increased endothelial permeability and defective repair are the hallmarks of several vascular diseases, including acute lung injury. However, little is known about the intrinsic pathways activating the endothelial cell (EC) regenerative programs. Objective: Studies have invoked a crucial role of S1P (sphingosine-1-phosphate) in resolving endothelial hyperpermeability through the activation of the GPCR (G-protein–coupled receptor), S1PR1 (S1P receptor 1). Here, we addressed mechanisms of generation of a population of S1PR1 ⁺ EC and their pivotal role in restoring endothelial integrity. Methods and Results: Studies were made using inducible EC-S1PR1 ^−/− ( iEC-S1PR1 ^−/− ) mice and S1PR1-GFP (green fluorescent protein) reporter mice to trace the generation of S1PR1 ⁺ EC. We observed in a mouse model of endotoxemia that S1P generation induced the programming of S1PR1 ^lo to S1PR1 ⁺ EC, which eventually comprised 80% of the lung EC. The cell transition was required for reestablishing the endothelial junctional barrier. We observed that conditional deletion of S1PR1 in EC increased endothelial permeability. RNA-seq analysis of S1PR1 ⁺ EC showed enrichment of genes regulating S1P synthesis and transport, specifically SPHK1 (sphingosine kinase 1) and SPNS2 (sphingolipid transporter 2). Activation of transcription factors EGR1 (early growth response 1) and STAT3 (signal transducer and activator of transcription 3) was required for transcribing SPHK1 and SPNS2, respectively, and both served to increase S1P production and amplify S1PR1 ⁺ EC transition. Furthermore, transplantation of S1PR1 ⁺ EC population into injured lung vasculature restored endothelial integrity. Conclusions: Our findings show that generation of the S1PR1 ⁺ EC population activates the endothelial regenerative program to mediate endothelial repair. Results raise the possibility of harnessing this pathway to restore vascular homeostasis in inflammatory vascular injury states.

Cell surface G protein–coupled receptors (GPCRs), upon agonist binding, undergo serine–threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

We have recently uncovered that endothelial cell (EC) S1PR1 controls the effectiveness of VEGFR2 driven tumor angiogenesis. By using tumor ECs, EC-S1PR1^−/- mice and S1PR1 antagonist, we showed that VEGF-VEGFR2 pathway requires EC-S1PR1-induced signaling to efficiently drive tumor vascularization and growth, indicating combining S1PR1 antagonist with anti-VEGF/VEGFR2 therapy may eradicate resistant tumors.

Open access peer-reviewed chapter