CHO adhesion on immobilized (10 mg/ml) fibronectin or (10 mg/ ml) vitronectin was determined in the presence or absence of the indicated concentrations of CXCL4 or CXCL4/CTF or CXCL4/ CTF-S as described in Material and Methods. Error bars represent the mean+SD, #, P,0.005; *, P,0.001 compared to the cell adhesion in the absence of CXCL4 or CXCL4/CTF; n = 2 independent experiments. Found at: doi:10.1371/journal.pone.0002657.s001 (39.08 MB DOC) Acknowledgments The authors thank Pr Martin A. Schwartz (University of Virginia, Charlottesville, United State) and Dr Serge Ahmed (Universite´ Victor- References 1. Folkman J (1995) Angiogenesis in cancer, vascular rheumatoid and other disease. Nat Med 1: 27–31. 2. Folkman J (1971) Tumor angiogensis: therapeutic implications. N Engl J Med 285: 1182–6. 3. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6: 273–86. 4. Levine SP, Wohl H (1976) Human p1atelet factor 4: Purification and characterization by affinity chromatography. J Biol Chem 51: 324–8. 5. Maione TE, Gray GS, Petro J, Hunt AJ, Donner AL, et al. (1990) Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science 247: 77–9. 6. Jouan V, Canron X, Alemany M, Caen JP, Quentin G, et al. (1999) Modulation of in vitro angiogenesis by platelet factor-4 derived peptides and mechanism of action. Blood 94: 984–93. 7. Hagedorn M, Zilberberg L, Lozano RM, Cuevas P, Canron X, et al. (2001) A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2. FASEB J 15: 550–2. 8. Sharpe RJ, Byers HR, Scott CF, Bauer SI, Maione TE (1990) Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4. J Natl Cancer Inst 82: 848–53. 9. Tanaka T, Manome Y, Wen P, Kufe DW, Fine HA (1997) Viral vector- mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat Med 3: 437–42. 10. Maione TE, Gray GS, Hunt AJ, Sharpe RJ (1991) Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity. Cancer Res 51: 2077–83. 11. Bello L, Giussani C, Carrabba G, Pluderi M, Lucini V, et al. (2002) Suppression of malignant glioma recurrence in a newly developed animal model by endogenous inhibitors. Clin Cancer Res 8: 3539–48. 12. Kolber DL, Knisely TL, Maione TE (1995) Inhibition of development of murine melanoma lung metastases by systemic administration of recombinant platelet factor 4. J Natl Cancer Inst 87: 304–9. 13. Davis GE, Senger DR (2005) Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res 97: 1093–107. 14. Brooks PC, Clark RA, Cheresh DA (1994a) Requirement of vascular integrin avb3 for angiogenesis. Science 264: 569–71. 15. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, et al. (1994b) Integrin avb3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79: 1157–64. 16. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, et al. (1995) Definition of two angiogenic pathways by distinct alpha v integrins. Science 270: 1500–2. 17. Kim S, Bell K, Mousa SA, Varner JA (2000) Regulation of angiogenesis in vivo by ligation of integrin a5b1 with the central cell-binding domain of fibronectin. Am J Pathol 156: 1345–62. 18. Fukami MH, Holmsen H, Kowalska MA, Niewiarowski S (2001) Platelet secretion. In: Colman RW, ed. Hemostasis and Thrombosis. Basis Principles and Clinical Practice. Philadelphia: Lippincott Williams & Wikins. pp 561– 573. 19. Hansell P, Maione TE, Borgstrom P (1995) Selective binding of platelet factor 4 to regions of active angiogenesis in vivo. Am J Physiol 269: 829–36. 20. Borgstrom P, Discipio R, Maione TE (1998) Recombinant platelet factor 4, an angiogenic marker for human breast carcinoma. Anticancer Res 18: 4035–41. 21. Schwartz MA, Schaller MD, Ginsberg MH (1995) Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol 11: 549–99. 22. Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA (1993) Integrin b1- and b3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol 121: 163–70. 23. Stromblad S, Becker JC, Yebra M, Brooks PC, Cheresh DA (1996) Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin avb3 during angiogenesis. J Clin Invest 98: 426–33. CXCL4 Integrins Angiogenesis Segalen Bordeaux 2) for critically reading the manuscript, Dr Alan Nurden (University Bordeaux II, France) for helpful discussion and Dr Francesca De Giorgi-Ichas, (INSERM U916-Institut Bergonie´, Bordeaux) for helpful technical assistance for confocal analysis. This work is dedicated to Aicha Mekki-Aidoudi and Bechir Aidoudi. Author Contributions Conceived and designed the experiments: SA AB. Performed the experiments: SA KB. Analyzed the data: SA AB. Contributed reagents/ materials/analysis tools: AB NK. Wrote the paper: SA AB. Designed the study: SA. 24. Hsu AR, Veeravagu A, Cai W, Hou LC, Tse V, et al. (2007) Integrin alpha (v)beta (3) antagonists for anti-angiogenic cancer treatment. Recent Patents Anticancer Drug Discov 2: 143–58. 25. Pasco S, Monboisse JC, Kieffer N (2000) The alpha 3(IV)185-206 peptide from noncollagenous domain 1 of type IV collagen interacts with a novel binding site on the beta 3 subunit of integrin avb3 and stimulates focal adhesion kinase and phosphatidylinositol 3-kinase phosphorylation. J Biol Chem 275: 32999–33007. 26. Humphries MJ (1996) Integrin activation: the link between ligand binding and signal transduction. Curr Opin Cell Biol 8: 632–40. 27. Rehn M, Veikkola T, Kukk-Valdre E, Nakamura H, Ilmonen M, et al. (2001) Interaction of endostatin with integrins implicated in angiogenesis. Proc Natl Acad Sci USA 98: 1024–1029. 28. Trikha M, Zhou Z, Timar J, Raso E, Kennel M, et al. (2002) Multiple roles for platelet GPIIb/IIIa and avb3 integrins in tumor growth, angiogenesis, and metastasis. Cancer Research 62: 2824–2833. 29. Pampoori N, Hato T, Stupack DG, Aidoudi S, Cheresh DA, et al. (1999) Mechanisms and consequences of affinity modulation of integrin avb3 detected with a novel patch-enginered monovalent ligand. J Biol Chem 274: 21609–21616. 30. Vuori K, Ruoslahti E (1993) Activation of protein kinase C precedes a5b1 integrin-mediated cell spreading on fibronectin. J Biol Chem 268: 21459–62. 31. Mould AP, Akiyama SK, Humphries MJ (1995) Regulation of integrin a5b1- fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J Biol Chem 270: 26270–7. 32. Lozano RM, Redondo-Horcajo M, Jimenez MA, Zilberberg L, Cuevas P, et al. (2001) Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity. J Biol Chem 276: 35723–34. 33. Yoshida A, Anand-Apte B, Zetter BR (1996) Differential Endothelial migration and proliferation to basic fibroblast growth factor and vascular endothelial growth factor. Growth Factors 13: 57–64. 34. Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, et al. (2000) A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 6: 851–60. 35. Sudhakar A, Nyberg P, Keshamouni VG, Mannam AP, Li J, et al. (2005) Human alpha1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by a1b1 integrin. J Clin Invest 115: 2801–10. 36. Magnon C, Galaup A, Mullan B, Rouffiac V, Bouquet C, et al. (2005) Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with avb3 and avb5 integrins. Cancer Res 65: 4353–61. 37. Sudhakar A, Sugimoto H, Yang C, Lively J, Zeisberg L, et al. (2003) Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by avb3 and a5b1 integrins. Proc Natl Acad Sci USA 100: 4766–71. 38. Maeshima Y, Sudhakar A, Lively JC, Ueki K, Kharbanda S, et al. (2002) Tumstatin, an endothelial cell-specific inhibitor of protein synthesis. Science 295: 140–3. 39. Koivunen E, Gay DA, Ruoslahti E (1993) Selection of peptides binding to the a5b1 integrin from phage display library. J Biol Chem 268: 20205–10. 40. Koivunen E, Wang B, Ruoslahti E (1994) Isolation of a highly specific ligand for the a5b1 integrin from a phage display library. J Cell Biol 124: 373–80. 41. Arap W, Pasqualini R, Ruoslahti E (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279: 377–80. 42. Felding-Habermann B, O’Toole TE, Smith J, Fransvea E, Ruggeri Z, et al. (2001) Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci USA 98: 1853–8. 43. De S, Razorenova O, McCabe NP, O’Toole T, Qin J, Byzova TV (2005) VEGF-integrin interplay controls tumor growth and vascularization. Proc Natl Acad Sci U S A 102: 7589–94. 44. Short SM, Derrien A, Narsimhan RP, Lawler J, Ingber DE, et al. (2005) Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by b1 integrins. J Cell Biol 168: 643–53. PLoS ONE | www.plosone.org 13 July 2008 | Volume 3 | Issue 7 | e2657