Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium–derived factor


Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium–derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium–derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.

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Figure 1: The roles of Fas and FasL induced by TSP1 and PEDF in apoptosis of cultured endothelial cells.
Figure 2: TSP1 and PEDF increased FasL mRNA and surface protein in endothelial cells.
Figure 3: Several angiogenic stimuli enhanced endothelial cell apoptosis by inhibitory TSP1, and increased surface Fas.
Figure 4: Expression of Fas receptor on newly forming capillaries induced in vivo.
Figure 5: Inactivation of Fas or FasL blocked the anti-angiogenic activity of TSP1 and PEDF in vitro and in vivo.


  1. 1

    Eliceiri, B.P. & Cheresh, D.A. The role of α-v-integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Invest. 103, 1227–1230 (1999).

    CAS  PubMed Central  Google Scholar 

  2. 2

    Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nature Med. 6, 389–395 (2000).

    CAS  PubMed Central  Google Scholar 

  3. 3

    Dejana, E., Bazzoni, G. & Lampugnani, M.G. Vascular endothelial (VE)-cadherin: only an intercellular glue? Exp. Cell Res. 252, 13–19 (1999).

    CAS  Google Scholar 

  4. 4

    Lucas, R. et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 92, 4730–4741 (1998).

    CAS  Google Scholar 

  5. 5

    Dhanabal, M. et al. Endostatin induces endothelial cell apoptosis. J Biol. Chem. 274, 11721–11726 (1999).

    CAS  Google Scholar 

  6. 6

    Jimenez, B. et al. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nature Med. 6, 41–48 (2000).

    CAS  Google Scholar 

  7. 7

    Stellmach, V.V., Crawford, S.E., Zhou, W. & Bouck, N. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc. Natl. Acad. Sci. U.S.A. 98, 2593–2597 (2001).

    CAS  PubMed Central  Google Scholar 

  8. 8

    Yue, T.L. et al. 2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression. Mol. Pharmacol. 51, 951–962 (1997).

    CAS  Google Scholar 

  9. 9

    Roberts, D.D. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J. 10, 1183–1191 (1996).

    CAS  Google Scholar 

  10. 10

    Becerra, S.P. Structure-function studies on PEDF. A non-inhibitory serpin with neurotrophic activity. Adv. Exp. Med. Biol. 425, 223–237 (1997).

    CAS  Google Scholar 

  11. 11

    Volpert, O.V., Lawler, J. & Bouck, N.P. A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1. Proc. Natl. Acad. Sci. U.S.A. 95, 6343–6348 (1998).

    CAS  PubMed Central  Google Scholar 

  12. 12

    Dawson, D. et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285, 245–248 (1999).

    CAS  Google Scholar 

  13. 13

    Mori, K. et al. Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J. Cell. Physiol. 188, 253–263 (2001).

    CAS  Google Scholar 

  14. 14

    Alberdi, E., Aymerich, M.S. & Becerra, S.P. Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor. J. Biol. Chem. 274, 31605–31612 (1999).

    CAS  Google Scholar 

  15. 15

    Bouck, N., Stellmach, V. & Hsu, S.C. How tumors become angiogenic. Adv. Cancer Res. 69, 135–174 (1996).

    CAS  Google Scholar 

  16. 16

    Benjamin, L.E. The controls of microvascular survival. Cancer Metastasis Rev. 19, 75–81 (2000).

    CAS  Google Scholar 

  17. 17

    Volpert, O.V. Modulation of endothelial cell survival by an inhibitor of angiogenesis thrombospondin-1: a dynamic balance. Cancer Metastasis Rev. 19, 87–92 (2000).

    CAS  Google Scholar 

  18. 18

    Golstein, P. Signal transduction. FasL binds preassembled Fas. Science 288, 2328–2329 (2000).

    CAS  Google Scholar 

  19. 19

    Bossi, G., Stinchcombe, J.C., Page, L.J. & Griffiths, G.M. Sorting out the multiple roles of Fas ligand. Eur. J. Cell Biol. 79, 539–543 (2000).

    CAS  Google Scholar 

  20. 20

    Krammer, P.H. CD95's deadly mission in the immune system. Nature 407, 789–795 (2000).

    CAS  Google Scholar 

  21. 21

    Okada, Y. et al. Reduced expression of flice-inhibitory protein (FLIP) and NFκB is associated with death receptor-induced cell death in human aortic endothelial cells (HAECs). Cytokine 15, 66–74 (2001).

    CAS  Google Scholar 

  22. 22

    Suhara, T., Mano, T., Oliveira, B. & Walsh, K. Phosphatidylinositol 3-kinase/Akt signaling controls endothelial cell sensitivity to Fas-mediated apoptosis via regulation of FLICE-inhibitory protein (FLIP). Circ. Res. 89, 13–19 (2001).

    CAS  Google Scholar 

  23. 23

    Jimenez, B. et al. c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1. Oncogene 20, 3443–3448 (2001).

    CAS  Google Scholar 

  24. 24

    Suri, C. et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171–1180 (1996).

    CAS  Google Scholar 

  25. 25

    Holash, J., Wiegand, S.J. & Yancopoulos, G.D. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 18, 5356–5362 (1999).

    CAS  PubMed Central  Google Scholar 

  26. 26

    Carmeliet, P. Basic concepts of (myocardial) angiogenesis: role of vascular endothelial growth factor and angiopoietin. Curr. Interv. Cardiol. Rep. 1, 322–335 (1999).

    CAS  Google Scholar 

  27. 27

    Gerber, H.P., Dixit, V. & Ferrara, N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J. Biol. Chem. 273, 13313–13316 (1998).

    CAS  Google Scholar 

  28. 28

    Tran, J. et al. Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem. Biophys. Res. Commun. 264, 781–788 (1999).

    CAS  PubMed Central  Google Scholar 

  29. 29

    Nor, J.E., Christensen, J., Mooney, D.J. & Polverini, P.J. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am. J. Pathol. 154, 375–384 (1999).

    CAS  PubMed Central  Google Scholar 

  30. 30

    O'Connor, D.S. et al. Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am. J. Pathol. 156, 393–398 (2000).

    CAS  PubMed Central  Google Scholar 

  31. 31

    Aoudjit, F. & Vuori, K. Matrix attachment regulates Fas-induced apoptosis in endothelial cells. A role for c-FLIP and implications for anoikis. J. Cell Biol. 152, 633–644 (2001).

    CAS  PubMed Central  Google Scholar 

  32. 32

    Freyberg, M.A., Kaiser, D., Graf, R., Buttenbender, J. & Friedl, P. Proatherogenic flow conditions initiate endothelial apoptosis via thrombospondin-1 and the Integrin-Associated Protein. Biochem. Biophys. Res. Commun. 286, 141–149 (2001).

    CAS  Google Scholar 

  33. 33

    Filleur, S. et al. In vivo mechanisms by which tumors producing thrombospondin 1 bypass its inhibitory effects. Genes Dev. 15, 1373–1382 (2001).

    CAS  PubMed Central  Google Scholar 

  34. 34

    Yeh, W.C. et al. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279, 1954–1958 (1998).

    CAS  Google Scholar 

  35. 35

    Varfolomeev, E.E. et al. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 9, 267–276 (1998).

    CAS  Google Scholar 

  36. 36

    Kaplan, H.J., Leibole, M.A., Tezel, T. & Ferguson, T.A. Fas ligand (CD95 ligand) controls angiogenesis beneath the retina. Nature Med. 5, 292–297 (1999).

    CAS  Google Scholar 

  37. 37

    Dawson, D.W. et al. CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J. Cell Biol. 138, 707–717 (1997).

    CAS  PubMed Central  Google Scholar 

  38. 38

    Sata, M. & Walsh, K. TNFα regulation of Fas ligand expression on the vascular endothelium modulates leukocyte extravasation. Nature Med. 4, 415–420 (1998).

    CAS  Google Scholar 

  39. 39

    Larsson, H. et al. Antiangiogenic effects of latent antithrombin through perturbed cell-matrix interactions and apoptosis of endothelial cells. Cancer Res. 60, 6723–6729 (2000).

    CAS  Google Scholar 

  40. 40

    Shichiri, M. & Hirata, Y. Antiangiogenesis signals by endostatin. FASEB J. 15, 1044–1053 (2001).

    CAS  Google Scholar 

  41. 41

    Bennett, M. et al. Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science 282, 290–293 (1998).

    CAS  Google Scholar 

  42. 42

    Sodeman, T., Bronk, S.F., Roberts, P.J., Miyoshi, H. & Gores, G.J. Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G992–G999 (2000).

    CAS  Google Scholar 

  43. 43

    Fukuo, K. et al. Activated T cells induce up-regulation of Fas antigen in cultured endothelial cells. Heart Vessels Suppl. 12, 81–83 (1997).

    Google Scholar 

  44. 44

    Sata, M., Suhara, T. & Walsh, K. Vascular endothelial cells and smooth muscle cells differ in expression of Fas and Fas ligand and in sensitivity to Fas ligand-induced cell death: implications for vascular disease and therapy. Arterioscler. Thromb. Vasc. Biol. 20, 309–316 (2000).

    CAS  Google Scholar 

  45. 45

    Kenyon, B.M. et al. A model of angiogenesis in the mouse cornea. Invest. Ophthalmol. Vis. Sci. 37, 1625–1632 (1996).

    CAS  Google Scholar 

  46. 46

    Passaniti, A. et al. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab. Invest. 67, 519–528 (1992).

    CAS  Google Scholar 

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We thank M. Wu for technical help. This work was supported by NIH grants RO1 CA52750 and CA64239 (N.B.), American Heart Association grant SDG0030023N, American Cancer Society grant RSG-01-099-01-CSM and NIH RO1 68003-01 (O.V.), Foundation for Fighting Blindness grants EY12826 (T.A.F.) and EY12707 (P.M.S.).

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Correspondence to Olga V. Volpert.

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Volpert, O., Zaichuk, T., Zhou, W. et al. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium–derived factor. Nat Med 8, 349–357 (2002).

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