Introduction: Septic hypercirculation is characterized by an increase in fluid extravastation accompanied by a loss of pericytes resulting in systemic hypotension and high mortality. The sphingolipid Sphingosine 1-phosphate has been shown to strengthen the connection between endothelial cells as well as endothelial cells and pericytes via trafficking of adhesion proteins (VE-Cadherin and N-Cadherin) to sites of cell-cell contacts and stabilizing the actin cytoskeleton. Furthermore, MRTF-A – an actin binding protein – has also been shown to play a vital role in endothelial-pericyte interactions via enchanced expression of SRF target genes, namely CCN1 and CCN2. In the vascular compartment, CCN1 and CCN2 promote the maturation of newly formed vessels by enhancing the pericyte coverage of endothelial cells in vivo. Nuclear translocation of MRTF-A is crucial in this signaling cascade and is dependent on the dissociation of MRTF-A from cytoplasmic g-actin. In this study we investigate the potential of S1P in preventing septic hypercirculation and the interplay between S1P, the stabilization of F-actin fibers and the subsequent enhancement of MRTF-A nuclear translocation and signaling.

Methods: C57Bl/6 mice were pretreated with S1P or PBS 12 hours before induction of sepsis via intraperitoneal injection of LPS (20mg/kg body weight). 6 hours after sepsis induction, vascular permeability was assessed using intravital microscopy to measure TRITC-dextrane extravasation. Furthermore, blood pressure was measured using a non-invasive blood measurement system for rodents (CODA 2, Kent Scientific). Survival was recorded for 96 hours. In a cell culture model, endothelial cells were stimulated with LPS and S1P followed by the assessment of Cadherin localization, actin cytoskeletal rearrangement and MRTF-A nuclear translocation.

Results: The treatment of septic mice with S1P drastically reduced the pericyte loss and capillary rarification regularily seen during systemic inflammation, an effect that was accompanied by a decrease in septic hyperpermeability as well as septic hypotension (MAP: PBS: 44,73 ± 3,36 vs. S1P: 69,98 ± 6,66 mmHg) and ultimately an increase in survival in septic mice (from 0% in control mice to 30% in S1P treated animals). Furthermore, S1P reduces VE-Cadherin phosphorylation and endothelial activation and additionally, S1P stabilizes filamentous actin and reduces the pool of cytoplasmic globular actin, thus facilitating the translocation of MRTF-A into the nucleus of endothelial cells, where it promotes pericyte coverage via CCN1 and CCN2, an effect demonstrated in vitro as well as in vivo.

Taken together these findings establish a potential signaling mechanism, in which S1P promotes SRF target gene expression via enhancing the nuclear availability of MRTF-A and thus the expression of the vessel stabilizing agents CCN1 and CCN2 during sepsis.