Sphingosine-1-phosphate-induced release of TIMP-2 from vascular smooth muscle cells inhibits angiogenesis Keith S. Mascall 1 , Gary R. Small 2, *, George Gibson 2 and Graeme F. Nixon 1,` 1 School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK 2 Department of Cardiothoracic Surgery, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, AB25 2ZD, UK *Present address: Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada K1Y 4W7 ` Author for correspondence (g.f.nixon@abdn.ac.uk) Accepted 3 January 2012 Journal of Cell Science 125, 2267–2275 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.099044 Summary Following myocardial infarction, angiogenesis occurs as a result of thrombus formation, which permits reperfusion of damaged myocardium. Sphingosine 1-phosphate (S1P) is a naturally occurring lipid mediator released from platelets and is found in high concentrations at sites of thrombosis. S1P might therefore be involved in regulating angiogenesis following myocardial infarction and might influence reperfusion. The aims of this study were to determine the effects of S1P in human coronary arterial cell angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-culture of human coronary artery endothelial cells, human coronary smooth muscle cells and human fibroblasts. In this model, S1P inhibited angiogenesis and this was dependent on the presence of smooth muscle cells. The mechanism of the inhibitory effect was through S1P-induced release of a soluble mediator from smooth muscle cells. This mediator was identified as tissue inhibitor of metalloproteinase-2 (TIMP-2). Release of TIMP- 2 was dependent on S1P-induced activation of Rho kinase and directly contributed to incomplete formation of endothelial cell adherens junctions. This was observed as a diffuse localisation of VE-cadherin, leading to decreased tubulogenesis. A similar inhibitory response to S1P was demonstrated in an ex vivo human arterial model of angiogenesis. In summary, S1P-induced inhibition of angiogenesis in human artery endothelial cells is mediated by TIMP-2 from vascular smooth muscle cells. This reduces the integrity of intercellular junctions between nascent endothelial cells. S1P might therefore inhibit the angiogenic response following myocardial infarction. Key words: Angiogenesis, Sphingosine 1-phosphate, Vascular smooth muscle, Metalloproteinase Introduction Angiogenesis is the growth of new blood vessels from the existing vasculature (Carmeliet and Jain, 2011). It is a key process in many (patho)physiological conditions and is tightly regulated. Angiogenic signals induce endothelial cells (ECs) to undergo a phenotypic change resulting in cellular proliferation and migration. This leads to the formation of sprouts from the existing blood vessels (Nicosia, 2009). Following growth of a sprout outward from the vessel, a complex set of cues leads to reformation of specialised endothelial–endothelial cell junctions (adherens junctions) (Carmeliet and Jain, 2011). Ultimately, lumen formation (tubulogenesis) occurs, although this process is still not clearly understood (Xu and Cleaver, 2011). The signals that regulate angiogenesis vary depending on the cellular environment, although typically, the growth factor vascular endothelial growth factor (VEGF) has a major role in most angiogenic scenarios. VEGF expression is induced by hypoxia, which is an important angiogenic stimulus (Fong, 2009). Sphingolipids, which are produced predominantly from sphingomyelin metabolism, are now known to have important effects on blood vessel function and might have a regulatory role in angiogenesis (Peters and Alewijnse, 2007; Lucke and Levkau, 2010). In particular, sphingosine 1-phosphate (S1P), a bioactive sphingolipid mediator present in plasma and released by activated platelets, can regulate vascular processes including motility and contractility, vascular proliferation and endothelial barrier permeability (Yatomi et al., 1995; Coussin et al., 2002; Kluk and Hla, 2001; Wang and Dudek, 2009). At a cellular level, S1P-induced effects are mediated through seven-transmembrane, G-protein-coupled receptors, S1P1–S1P5 (Meyer zu Heringdorf and Jakobs, 2007). These receptors activate a repertoire of intracellular signalling pathways (Sanchez and Hla, 2004). S1P1, through the heterotrimeric G-protein Gi, leads to activation of several pathways including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). S1P2 and S1P3 couple to G12 or G13, and Gq, respectively, and predominantly activate intracellular Ca 2+ release and the monomeric G-protein RhoA (Rosen et al., 2009). Most cell types in the vasculature express at least two isoforms of S1P1, S1P2 or S1P3. In cardiovascular disease, angiogenesis occurs following myocardial infarction (MI) (van der Laan et al., 2009). This process can lead to an enhanced reperfusion of damaged myocardium and might be therapeutically beneficial. One major stimulus for angiogenesis in this case is probably the hypoxia that results from thrombus formation and subsequent disruption in blood supply to the heart. This hypoxia leads to an upregulation of hypoxia-inducible transcription factors resulting in increased expression of angiogenic growth factors such as Research Article 2267 Journal of Cell Science