differentiation of mesoangioblasts towards a SM phenotype. contractile Supporting Information Table S1 Genes up-regulated by S1P treatment in human mesoangioblasts (DOC) References 1. Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801. 2. Owens GK (1995) Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75: 487–517. 3. Brunelli S, Tagliafico E, De Angelis FG, Tonlorenzi R, Baesso S, et al. (2004) Msx2 and necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells. Circ Res 94: 1571–1578. 4. Brunelli S, Cossu G (2005) A role for MSX2 and necdin in smooth muscle differentiation of mesoangioblasts and other mesoderm progenitor cells. Trends Cardiovasc Med 15: 96–100. 5. Han CI, Campbell GR, Campbell JH (2001) Circulating bone marrow cells can contribute to neointimal formation. J Vasc Res 38: 113–119. 6. Minasi MG, Riminucci M, De Angelis L, Borello U, Berarducci B, et al. (2002) The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development 129: 2773–2783. 7. Galli D, Innocenzi A, Staszewsky L, Zanetta L, Sampaolesi M, et al. (2005) Mesoangioblasts, vessel-associated multipotent stem cells, repair the infarcted heart by multiple cellular mechanisms: a comparison with bone marrow progenitors, fibroblasts, and endothelial cells. Arterioscler Thromb Vasc Biol 25: 692–697. 8. Pyne S, Pyne NJ (2000) Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 349: 385–402. 9. Daum G, Grabski A, Reidy MA (2009) Sphingosine 1-Phosphate. A Regulator of Arterial Lesions. Arterioscler Thromb Vasc Biol 29: 1439–1443. 10. Pebay A, Bonder CS, Pitson SM (2007) Stem cell regulation by lysopho- spholipids. Prostaglandins Other Lipid Mediat 84: 83–97. 11. Donati C, Cencetti F, Nincheri P, Bernacchioni C, Brunelli S, et al. (2007) Sphingosine 1-phosphate mediates proliferation and survival of mesoangioblasts. Stem Cells 25: 1713–1719. 12. Donati C, Cencetti F, De Palma C, Rapizzi E, Brunelli S, et al. (2009) TGFbeta protects mesoangioblasts from apoptosis via sphingosine kinase-1 regulation. Cell Signal 21: 228–236. 13. Davis MD, Clemens JJ, Macdonald TL, Lynch KR (2005) Sphingosine 1- phosphate analogs as receptor antagonists. J Biol Chem 280: 9833–9841. 14. Osada M, Yatomi Y, Ohmori T, Ikeda H, Ozaki Y (2002) Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist. Biochem Biophys Res Commun 299: 483–487. 15. Mitra P, Oskeritzian CA, Payne SG, Beaven MA, Milstien S, et al. (2006) Role of ABCC1 in export of sphingosine-1-phosphate from mast cells. Proc Natl Acad Sci U S A 103: 16394–16399. 16. Morosetti R, Mirabella M, Gliubizzi C, Broccolini A, De Angelis L, et al. (2006) MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle. Proc Natl Acad Sci U S A 103: 16995–17000. 17. Nincheri P, Bernacchioni C, Cencetti F, Donati C, Bruni P (2010) Sphingosine kinase-1/S1P(1) signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts. Cell Signal 22: 1688–1699. 18. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116–5121. 19. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80. 20. Nincheri P, Luciani P, Squecco R, Donati C, Bernacchioni C, et al. (2009) Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells. Cell Mol Life Sci 66: 1741–1754. 21. Donati C, Meacci E, Nuti F, Becciolini L, Farnararo M, et al. (2005) Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor. FASEB J 19: 449–451. 22. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(2Delta Delta C(T)) Method. Methods 25: 402–408. 23. D’Alessio S, Fibbi G, Cinelli M, Guiducci S, Del Rosso A, et al. (2004) Matrix metalloproteinase 12-dependent cleavage of urokinase receptor in systemic S1P Differentiates Mesoangioblasts Table S2 Genes down-regulated by S1P treatment in human mesoangioblasts (DOC) Author Contributions Conceived and designed the experiments: CD PB. Performed the experiments: CD GM SS FC CB GN. Analyzed the data: CD AM MB PB. Contributed reagents/materials/analysis tools: SB GC. Wrote the paper: CD PB. Financial support: PB FT CD. sclerosis microvascular endothelial cells results in impaired angiogenesis. Arthritis Rheum 50: 3275–3285. 24. Willis SN, Adams JM (2005) Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol 17: 617–625. 25. Bergamaschi D, Samuels Y, Sullivan A, Zvelebil M, Breyssens H, et al. (2006) iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72-polymorphic p53. Nat Genet 38: 1133–1141. 26. Narita N, Heikinheimo M, Bielinska M, White RA, Wilson DB (1996) The gene for transcription factor GATA-6 resides on mouse chromosome 18 and is expressed in myocardium and vascular smooth muscle. Genomics 36: 345–348. 27. Suzuki E, Evans T, Lowry J, Truong L, Bell DW, et al. (1996) The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals. Genomics 38: 283–290. 28. Lepore JJ, Cappola TP, Mericko PA, Morrisey EE, Parmacek MS (2005) GATA-6 regulates genes promoting synthetic functions in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 25: 309–314. 29. Malik TH, Shoichet SA, Latham P, Kroll TG, Peters LL, et al. (2001) Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1. EMBO J 20: 1715–1725. 30. Rath N, Wang Z, Lu MM, Morrisey EE (2005) LMCD1/Dyxin is a novel transcriptional cofactor that restricts GATA6 function by inhibiting DNA binding. Mol Cell Biol 25: 8864–8873. 31. Kanematsu A, Ramachandran A, Adam RM (2007) GATA-6 mediates human bladder smooth muscle differentiation: involvement of a novel enhancer element in regulating alpha-smooth muscle actin gene expression. Am J Physiol Cell Physiol 293: C1093–C1102. 32. Radeke HH, von Wenckstern H, Stoidtner K, Sauer B, Hammer S, et al. (2005) Overlapping signaling pathways of sphingosine 1-phosphate and TGF-beta in the murine Langerhans cell line XS52. J Immunol 174: 2778–2786. 33. Xin C, Ren S, Kleuser B, Shabahang S, Eberhardt W, et al. (2004) Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transform- ing growth factor-beta-induced cell responses. J Biol Chem 279: 35255–35262. 34. Lebman DA, Spiegel S (2008) Cross-talk at the crossroads of sphingosine-1- phosphate, growth factors, and cytokine signaling. J Lipid Res 49: 1388–1394. 35. Watterson KR, Lanning DA, Diegelmann RF, Spiegel S (2007) Regulation of fibroblast functions by lysophospholipid mediators: potential roles in wound healing. Wound Repair Regen 15: 607–616. 36. Tagliafico E, Brunelli S, Bergamaschi A, De Angelis L, Scardigli R, et al. (2004) TGFbeta/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts. J Cell Sci 117: 4377–4388. 37. Lockman K, Hinson JS, Medlin MD, Morris D, Taylor JM, et al. (2004) Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors. J Biol Chem 279: 42422–42430. 38. Wamhoff BR, Lynch KR, Macdonald TL, Owens GK (2008) Sphingosine-1- phosphate receptor subtypes differentially regulate smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol 28: 1454–1461. 39. Medlin MD, Staus DP, Dubash AD, Taylor JM, Mack CP (2010) Sphingosine 1- phosphate receptor 2 signals through leukemia-associated RhoGEF (LARG), to promote smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 30: 1779–1786. 40. Grabski AD, Shimizu T, Deou J, Mahoney WM, Jr., Reidy MA, et al. (2009) Sphingosine-1-phosphate receptor-2 regulates expression of smooth muscle alpha-actin after arterial injury. Arterioscler Thromb Vasc Biol 29: 1644–1650. 41. Cencetti F, Bernacchioni C, Nincheri P, Donati C, Bruni P (2010) Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis. Mol Biol Cell 21: 1111–1124. 42. Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, et al. (2000) Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest 106: 951–961. 43. Pebay A, Wong RC, Pitson SM, Wolvetang EJ, Peh GS, et al. (2005) Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 23: 1541–1548. 44. Sachinidis A, Gissel C, Nierhoff D, Hippler-Altenburg R, Sauer H, et al. (2003) Identification of plateled-derived growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell Physiol Biochem 13: 423–429. PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20389