Abstract 14973: Upregulated Sphingosine-1-Phosphate Receptor 3 Disrupts Endothelial Cell Barrier and Mitochondrial Network Dynamics

Introduction: Ventilator-Induced Lung Injury (VILI) exacerbates existing Acute Lung Injury (ALI) of ICU patients via mechanical ventilation. However, the cellular and molecular mechanism behind VILI is still poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive lipid which usually enhances vascular endothelial barrier via S1P receptor 1 (S1PR1) to protect against ALI. VILI associated mechanical stress reduces S1PR1 while increases S1P receptor 3 (S1PR3) expression. S1PR3 couples to Rho, a central regulator of cytoskeleton and mitochondrial network dynamics. Therefore, the upregulation of S1P-S1PR3 pathway is highly likely to be involved in the deterioration of endothelial barrier and mitochondrial function during VILI. Methods and Results: Cyclic stretch (18%) significantly increased S1PR3 levels (1.85±0.12 fold, n=3, p<0.05) in human lung endothelial cells (EC). After S1PR3 overexpression (S1PR3 OE), EC basal resistance was reduced (62.8±4.3%, n=8, p<0.05) in ECIS experiments. Moreover, S1P (100 nM) increased 37.2±4.1% resistance in control cells (n=5, p<0.05), while decreased 32.0±3.2% resistance in S1PR3 OE cells (n=5, p<0.05). In contrast to S1P-induced junctional distribution of VE-cadherin in control cells, S1P failed to cause any VE-cadherin junctional redistribution in S1PR3 OE cells as shown by Immunofluorescence (Fig. 1). We further confirmed that S1PR3 OE suppresses VE-cadherin expression in human ECs by western blot. In addition, in S1PR3 OE cells, S1P challenge induced an increased mitochondrial ROS release as shown by MitoSOX assay (6.87±1.10 fold, n=3, p<0.05) and a mitochondrial fission phenotype that was not observed in control cells. Conclusions: VILI associated mechanical stress upregulates S1PR3, leading to the reprograming of endothelial cell cytoskeleton regulatory machinery and mitochondrial network dynamics. S1PR3 might be a promising target to ameliorate the damage caused by mechanical ventilation.