Fingolimod does not prevent syndecan-4 shedding from the endothelial glycocalyx in a cultured human umbilical vein endothelial cell model of vascular injury

Abstract

Background: Shedding of the endothelial glycocalyx (EG) is associated with poor outcomes in a range of conditions including sepsis. Fresh frozen plasma (FFP) restores the damaged EG to baseline thickness, however the mechanism for this effect is unknown, and some components of FFP have adverse effects unrelated to the EG. There is some limited evidence that sphingosine-1-phosphate (S1P) within FFP restores the EG by activating the endothelial cell S1P receptor 1 (S1PR₁). However, there are disadvantages to using S1P clinically as an EG restorative therapy. A potential alternative is the S1PR agonist fingolimod (FTY720). The aim of this study was to assess whether FTY720 prevents EG shedding in injured cultured human umbilical vein endothelial cells.

Methods: Shedding of the EG was induced in cultured human umbilical vein endothelial cells (HUVECs) by exposure to adrenaline, TNF-α and H₂O₂. The cells were then assigned to one of six conditions for 4 h: uninjured and untreated, injured and untreated, injured and treated with FTY720 with and without the S1PR₁ inhibitor W146, and injured and treated with 25% FFP with and without W146. Syndecan-4, a component of the EG, was measured in cell supernatants, and syndecan-4 and thrombomodulin mRNA expression was quantitated in cell lysates.

Results: The injury resulted in a 2.1-fold increase in syndecan-4 (p < 0.001), consistent with EG shedding. Syndecan-4 and thrombomodulin mRNA expression was increased (p < 0.001) and decreased (p < 0.05), respectively, by the injury. Syndecan-4 shedding was not affected by treatment with FTY720, whereas FFP attenuated syndecan-4 shedding back to baseline levels in the injured cells and this was unaffected by W146. Neither treatment affected syndecan-4 or thrombomodulin mRNA expression.

Conclusions: FTY720 did not prevent syndecan-4 shedding from the EG in the HUVEC model of endothelial injury, suggesting that activation of S1PR does not prevent EG damage. FFP prevented syndecan-4 shedding from the EG via a mechanism that was independent of S1PR₁ and upregulation of SDC-4 production. Further studies to examine whether FTY720 or another S1PR agonist might have EG-protective effects under different conditions are warranted, as are investigations seeking the mechanism of EG protection conferred by FFP in this experimental model.

Keywords: Cell culture; Endothelium; FTY720; Fingolimod; Fresh frozen plasma; Glycocalyx; Human umbilical vein endothelial cells; Sphingosine 1-phosphate; Syndecan-1; Syndecan-4.

Fig. 1

Experimental protocol. Asterisk indicates injury conditions: 1.0 nM adrenaline, 10 ng/mL TNF-α, and 100 μM H2O2 in a 5% CO₂ incubator at 37 °C

Fig. 2

Syndecan-4 (SDC-4) concentration in cell culture supernatant. The effect of treatment with FTY720 or fresh frozen plasma (FFP) with and without 10 $\mathrm{\mu }$M of the sphingosine 1-phosphate receptor 1 (S1PR₁) antagonist W146 on SDC-4 shedding from injured cultured human umbilical vein endothelial cells (HUVECs). **p < 0.01, ***p < 0.001; N = 5 for each group

Fig. 3

Relative expression of syndecan-1 (SDC-1) mRNA. The effect of treatment with FTY720, with and without 10 $\mathrm{\mu }$M of the sphingosine 1-phosphate receptor 1 (S1PR₁) antagonist W146, or fresh frozen plasma (FFP) on the relative expression of SDC-1 mRNA from injured cultured human umbilical vein endothelial cells (HUVECs)

Fig. 4

Relative expression of glypican-1 (GP-1) mRNA. The effect of treatment with FTY720, with and without 10 $\mathrm{\mu }$ M of the sphingosine 1-phosphate receptor 1 (S1PR₁) antagonist W146, or fresh frozen plasma (FFP) on the relative expression of GP-1 mRNA from injured cultured human umbilical vein endothelial cells (HUVECs)

Fig. 5

Relative expression of thrombomodulin (TM) mRNA. The effect of treatment with FTY720, with and without 10 $\mathrm{\mu }$M of the sphingosine 1-phosphate receptor 1 (S1PR₁) antagonist W146, or fresh frozen plasma (FFP) on the relative expression of TM mRNA from injured cultured human umbilical vein endothelial cells (HUVECs). *p < 0.05, **p < 0.01, N = 5 for each group

Fig. 6

Relative expression of syndecan-4 (SDC-4) mRNA. The effect of treatment with FTY720, with and without 10 $\mathrm{\mu }$ M of the sphingosine 1-phosphate receptor 1 (S1PR₁) antagonist W146, or fresh frozen plasma on the relative expression of SDC-4 mRNA from injured cultured human umbilical vein endothelial cells (HUVECs). *p < 0.05, **p < 0.01, ***p < 0.001; N = 5 for each group