Sphingolipids inhibit angiogenesis via activation of the S1P2 receptor and Rho-kinase

Keith Mascall, Gary Small, Graeme Nixon. University of Aberdeen, Aberdeen, United Kingdom.

Sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are naturally occurring sphingolipids released from platelets and bound to lipoproteins. Both lipids act as extracellular ligands at the GPCR-family of S1P receptors which are expressed on endothelial cells. The aim of this study is to determine the effects of S1P and SPC on ex vivo and in vitro angiogenesis.

Mouse aortic rings were cultured on Matrigel with standard growth factors at 37°C and incubated with vehicle (BSA (4mg/ml in 10% DMSO)), S1P (1μM) or SPC (2μM). Tubule microvessel outgrowths branching from rings were counted. Data represents mean number of tubules formed ± sem at day 7 of growth. Both S1P and SPC significantly reduced tubule formation; 28±6 (n = 5) and 27±4 (n = 9) respectively in comparison to control 82±4 (n = 29) (p<0.05, one-way ANOVA). To examine potential mechanisms, rings were pre-incubated with the S1P2 receptor antagonist, JTE-013 (1μM). Incubation with JTE-013 significantly reversed the decrease in tubule formation produced by S1P (60±8) or SPC (59±9) (n = 10). As the S1P2 receptor is coupled to the RhoA/Rho-kinase pathway, rings were pre-incubated with the Rho-kinase inhibitor Y-27632 (10μM). Inhibition of Rho-kinase also significantly reversed the S1P- and SPC-induced decrease in tubule formation (S1P - 54±8; SPC - 52±10, n = 10).

A co-culture model of angiogenesis using primary cultured human fibroblasts, human coronary artery vascular smooth muscle cells and human coronary artery endothelial cells was also investigated. Co-cultures were incubated with either vehicle, S1P (1μM) or SPC (2μM). After 14 days, endothelial tubule formation was visualized by immunofluorescence labelling with anti-CD146 antibody. Fluorescent images were analysed using ImageJ software. S1P and SPC both significantly inhibited tubule formation in these triple layer cultures by 80±7% and 75±7% (n = 7). Addition of either JTE-013 or Y27632 significantly reversed this inhibition.

In conclusion, both S1P and SPC inhibit angiogenesis in ex vivo and in vitro models. This effect is partly via binding to the S1P2 receptor and subsequent activation of RhoA/Rho-kinase pathway.