Inflammatory Conditions Disrupt Constitutive Endothelial Cell Barrier Stabilization by Alleviating Autonomous Secretion of Sphingosine 1-Phosphate.

Jefri Jeya Paul,Sarah Spiegel,Cynthia Weigel,Tina Müller,Regine Heller,Markus H Gräler

doi:10.3390/cells9040928

Abstract

The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.

Highlights

Endothelial cell (EC) barriers are important intercellular structures that regulate the movement of fluids and dissolved substances into tissues
Quantitative PCR demonstrated that both, human umbilical vein endothelial cells (HUVEC) and EA.hy926 expressed mainly S1P receptor type 1 (S1PR1) followed by S1PR3, HUVEC expressed both receptors stronger than EA.hy926 (Figure 1A)
Specific staining was demonstrated by the incubation of cells with 1 μM FTY720 overnight, which led to S1PR1 internalization and low cell surface staining as expected (Figure 1B)

Summary

Introduction

Endothelial cell (EC) barriers are important intercellular structures that regulate the movement of fluids and dissolved substances into tissues. 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. 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

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Conclusion