Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC234

Poster Communications

Sphingosine 1-phosphate inhibits angiogenesis in human arteries

K. S. Mascall1, G. R. Small1, G. Gibson2, G. F. Nixon1

1. School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom. 2. Department of Cardiothoracic Surgery, University of Aberdeen, Aberdeen, United Kingdom.


The formation of new blood vessels by angiogenesis is a key pathophysiological process that can be activated in ischaemic conditions. The naturally occurring lipid mediator, sphingosine 1-phosphate (S1P), is released from activated platelets and at sites of thrombosis where ischaemia may result. S1P is an important regulator of the vasculature through activation of S1P receptors and could play a role in controlling new vessel formation and subsequent reperfusion of ischaemic tissues. In this study we aim to examine the effects of S1P on human arterial angiogenesis. The effects of pathological S1P concentrations on endothelial tubule formation were assessed by an ex vivo angiogenic ring model and an in vitro co-culture model. Human left internal mammary artery rings were cultured on Matrigel with standard growth factors at 37°C and incubated with vehicle or S1P (1µM). Tubule microvessel outgrowths branching from rings were counted. S1P significantly reduced mean tubule number formed at day 14 (22±3, n=14) compared to vehicle (63±9, n=20) (p<0.05, one-way ANOVA). To examine potential mechanisms, rings were pre-incubated with S1P2 receptor antagonist, JTE-013 (1µM). JTE-013 significantly reversed the decrease in tubule formation produced by S1P (n=7). As S1P2 receptors couple to the RhoA/Rho-kinase pathway, the effects of the Rho-kinase inhibitor Y27632 (10µM) were determined. Inhibition of Rho-kinase significantly reversed the S1P-induced decrease in tubule formation (44±3, n=7). 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 vehicle or S1P (1µM). After 14 days, endothelial tubule formation was visualized by immunofluorescence labelling with endothelial selective anti-CD146 antibody. Fluorescent images were analysed using ImageJ software. S1P significantly inhibited tubule formation in these co-cultures by 80±7% (n=7, p<0.05, ANOVA). The signalling mechanism responsible also occurred via S1P2 and Rho/Rho-kinase signalling, as addition of JTE-013 or Y27632 significantly reversed the S1P-induced inhibition. The potential mechanisms were further investigated by examining formation of adherens junctions. Expression of VE-cadherin, an essential junctional protein, was unchanged in endothelial cells following S1P incubation. Staining with anti-VE-cadherin antibody in co-cultures, however, revealed a diffuse localization in S1P-treated co-cultures which suggests a disruption to adherens junction formation. In conclusion, S1P inhibits angiogenesis in human arteries in an S1P2 dependent manner. This S1P-induced angiostatic effect occurred through a mechanism involving a disruption of endothelial cell-cell contact.

Where applicable, experiments conform with Society ethical requirements