Endothelial cell (EC) barriers are important intercellular structures that regulate the movement of fluids and dissolved substances into tissues. Maintaining barrier function is particularly important at sites where fluids need to be efficiently separated from tissues such as the vasculature, lymph vessels, gut, brain, and lung [1]. Several different junctional complexes are involved in barrier formation, including tight and adherens junctions, gap junctions, and desmosomes [2]. Adherens junctions are formed by cadherins and nectins and provide a mechanical linker similar to zippers, while tight junctions are formed by claudins, occludin, and junctional adhesion molecules in the transmembrane regions and perform most of the sealing functions to prevent passage of fluids and molecules [3,4]. The bioactive sphingolipid signaling molecule sphingosine 1-phosphate (S1P) and its G protein-coupled receptor S1PR1 are critical mediators of adherens junction assembly [5]. The deletion of S1PR1 in ECs or the deletion of the two known S1P-producing sphingosine kinases (SphK1 and SphK2) in hematopoietic cells and ECs of adult mice result in severe disruption of the EC barrier [6,7,8]. Despite this apparent phenotype, the exact mechanism of barrier maintenance by S1P is still unknown. One of the most puzzling questions is of how high S1P concentrations in circulation that are sufficient to induce activation-induced internalization and desensitization of S1PR1 are able to constitutively maintain the vascular EC barrier. Two models were proposed as potential explanations [8]: (1) the static model postulating that there is constantly sufficient S1PR1 expression on the luminal cell surface of ECs even at high S1P concentrations, due to efficient receptor recycling and (2) the dynamic model suggesting that S1PR1 is only expressed on the tissue-facing side of vascular ECs which are activated by S1P leaking through the EC barrier and subsequently induce adherens junction assembly and EC barrier stabilization. In either case, reduced S1P leakage and decreased barrier stability occur until the amount of S1P leaking through the EC barrier increases again and starts a new cycle of EC barrier formation. The validity of either of these models has not yet been verified.

The collapse of the EC barrier is a life-threatening condition and a major severity factor in sepsis [9]. The S1P concentration in circulation decreases significantly during systemic inflammation [10,11,12,13]. Whether or not this observed decrease of S1P has something to do with the vascular EC barrier collapse is not known. Previous data indicate that even low amounts of S1P in plasma are sufficient to maintain S1P and S1PR1 mediated lymphocyte circulation [14]. 

Here, we show that, in vitro, ECs can autonomously produce and secrete S1P, rendering their ability to maintain EC barrier formation largely independent from exogenously added S1P. The S1P transporter Spinster homolog 2 (Spns2) plays a crucial role in the proper release of S1P into the extracellular space. Our work has uncovered an important function of Spns2 in ECs to regulate barrier stability. Spns2 deficient mice demonstrated significantly reduced EC barrier formation presumably due to the lack of S1P exportation from ECs. Furthermore Spns2, but not S1PR1, was down-regulated in ECs stimulated with lipopolysaccharides (LPS) and pro-inflammatory cytokines. Inflammation-induced EC barrier breakdown due to down-regulation of Spns2 resulted in decreased S1P release. Thus, decreased exportation of S1P from ECs due to reduced expression of Spns2 may contribute to EC barrier dysfunction during inflammation. This mechanism may be particularly important in sepsis, where inflammation-induced collapse of the EC barrier significantly contributes to increased morbidity and mortality. The observed stable expression of S1PR1 and the most likely local autocrine and paracrine activity of Spns2-released S1P points to local approaches for S1P supplementation in tissues rather than systemic alteration of S1P in circulating plasma as a potential medical intervention.