Aortic valve stenosis (AVS) is closely associated with increased valve inflammation leading to calcification and ultimately results in cardiac failure if untreated. Currently the only therapeutic option is surgical valve replacement. Recent studies have indicated that increased angiogenesis within the valve may facilitate access of inflammatory cells and this plays an important role in the pathogenesis of AVS. Preventing angiogenesis in aortic valves is therefore a novel therapeutic target in this disease. Sphingosine 1-phosphate (S1P) is a lipid mediator present in plasma and produces intracellular effects via specific S1P receptors. We have previously shown that S1P can inhibit angiogenesis in human arteries (Mascall et al, 2012). The aim of the current study was to determine the effect of S1P on angiogenesis in human aortic valves. Valves from patients undergoing valve replacement surgery for AVS (diseased) or aortic regurgitation (non-diseased) were cut 2mm2 pieces and embedded in Matrigel. Valve pieces were incubated for up to 14 days in growth factor-supplemented medium and angiogenic sprouts were quantified. Sprouts consisting of endothelial cells (confirmed by uptake of acetylated-LDL) increased in number from day 5 until day 14 and this was significantly greater in diseased compared to non-diseased valves (number of sprouts observed at day 14, non-diseased 60 ± 8 versus diseased 110 ± 12, n=4 per group, mean ± s.e.m., ANOVA, p<0.05). Treatment of either diseased or non-diseased valve tissue with 1μM S1P resulted in a significant decrease in endothelial sprout formation compared to non-treated valves (number of sprouts observed at day 14, non-diseased valves – untreated 71 ± 5 versus S1P-treated 18 ± 3, n=3 per group; diseased valves – untreated 116 ± 9 versus S1P-treated 38 ± 5, n=3 per group, p<0.05). To examine this further, we assessed the activity of matrix metalloprotineases (MMPs) which are associated with angiogenesis. Activity of MMP-2 was significantly greater in diseased compared to control valves as assessed by in vitro gelatinolytic activity. Incubation with 1μM S1P decreased MMP activity in diseased valves (gelatinolytic activity observed at day 14, 94 ± 11% decrease in untreated valves compared to treated, n=3 per group, p<0.05). To determine the potential mechanisms involved we examined expression of the angiostatic protein, soluble fms-like tyrosine kinase-1 (sflt-1). Immunoblotting of valve homogenates demonstrated that sflt-1 expression is significantly decreased in diseased valves compared to control valves. Homogenates from diseased valves incubated with 1μM S1P for 14 days had a significantly increased expression of sflt-1 (n=3 per group). In conclusion, S1P can inhibit angiogenesis in human aortic valves, possibly via increasing expression of the angiostatic protein sflt-1. This mechanism may provide a therapeutic target in AVS.