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Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1

Nature Medicine volume 6, pages 41–48 (2000)Cite this article

Abstract

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.

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Figure 1: TSP-1-induced association of activated p59fyn with CD36 in human microvascular endothelial cells.
Figure 2: p38 MAP kinase activation by TSP-1 in microvascular endothelial cells and requirement for CD36 engagement, Fyn and caspases.
Figure 3: p38MAPK-dependent activation of caspase 3-like activity.
Figure 4: Prevention of TSP-1 repression of endothelial cell migration and TSP-1 induction of apoptosis by inhibition of p38MAPK and caspases.
Figure 5: In situ identification of apoptotic endothelial cells.
Figure 6: Signaling molecules induced by TSP-1 in microvascular endothelial cells essential for inhibition of angiogenesis.

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References

  1. Hanahan, D. & Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353–364 (1996).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. 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. USA 95, 6343–6348 (1998)

    Article  CAS  Google Scholar 

  4. O'Reilly, M.S. et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315–328 (1994).

    Article  CAS  Google Scholar 

  5. O'Reilly, M.S. et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277–285 (1997).

    Article  CAS  Google Scholar 

  6. Boehm, T., Folkman, J., Browder, T. & O'Reilly, M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404–407 ( 1997).

    Article  CAS  Google Scholar 

  7. Guo, N., Krutzsch, H.C., Inman, J.K. & Roberts, D.D. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res. 57, 1735–1742 (1997).

    CAS  PubMed  Google Scholar 

  8. Claesson-Welsh, L. et al. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc. Natl. Acad. Sci. USA 95, 5579– 5583 (1998).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. 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 (1992).

    Article  Google Scholar 

  12. DiPietro, L.A. in Regulation of Angiogenesis (eds. Goldberg. I.D. & Rosen, E.M.) 295–313 (Birkhauser Verlag, Basel, Switzerland, 1997).

    Book  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Dawson, D.W. & Bouck, N. in Antiangiogenic Agents in Cancer (ed. Teicher, B.A.) 185–204 (Humana, Totowa, New Jersey, 1998).

    Book  Google Scholar 

  15. Crawford, S.E. et al. Thrombospondin-1 is a major activator of TGF-β1 in vivo. Cell 93, 1159– 1170 (1998).

    Article  CAS  Google Scholar 

  16. Weinstat-Saslow, D.L. et al. Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 54, 6504–6511 (1994).

    CAS  PubMed  Google Scholar 

  17. Castle, V.P., Dixit, V.M. & Polverini, P.J. Thrombospondin-1 suppresses tumorigenesis and angiogenesis in serum-and anchorage-independent NIH 3T3 cells. Lab. Invest. 77, 51–61 (1997).

    CAS  PubMed  Google Scholar 

  18. Bleuel, K., Popp, S., Fusenig, N.E., Stanbridge, E.J. & Boukamp, P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization . Proc. Natl. Acad. Sci. USA 96, 2065– 2070 (1999).

    Article  CAS  Google Scholar 

  19. Volpert, O.V. et al. Inhibition of angiogenesis by thrombospondin-2. Biochem. Biophys. Res. Comm. 217, 326– 332 (1995).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Huang, M.M., Bolen, J.B., Barnwell, J.W., Shattil, S.J. & Brugge, J.S. Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. Proc. Natl. Acad. Sci. USA 88, 7844–7848 (1991).

    Article  CAS  Google Scholar 

  22. Bull, H.A., Brickell, P.M. & Dowd, P.M. Src-related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells. FEBS Lett. 351, 41– 44 (1994).

    Article  CAS  Google Scholar 

  23. Tolsma, S.S. et al. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J. Cell Biol. 122, 497–511 (1993).

    Article  CAS  Google Scholar 

  24. Cosulich, S. & Clarke, P. Apoptosis: does stress kill?. Curr. Biol. 6, 1586–1588 (1996).

    Article  CAS  Google Scholar 

  25. Le-Niculescu, H. et al. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death . Mol. Cell. Biol. 19, 751– 763 (1999).

    Article  CAS  Google Scholar 

  26. Verheij, M. et al. Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature 380, 75– 79 (1996).

    Article  CAS  Google Scholar 

  27. Ichijo, H. et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90–94 (1997).

    Article  CAS  Google Scholar 

  28. Kumar, S. et al. Activation of the HIV-1 long terminal repeat by cytokines and environmental stress requires an active CSBP/p38 MAP kinase. J. Biol. Chem. 271, 30864–30869 (1996).

    Article  CAS  Google Scholar 

  29. Polverini, P.J., Bouck, N.P. & Rastinejad, F. Assay and purification of naturally occurring inhibitor of angiogenesis. Meth. Enzymol. 198, 440 –450 (1991).

    Article  CAS  Google Scholar 

  30. Schultz-Cherry, S. & Murphy-Ullrich, J.E. Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J. Cell Biol. 122, 923–932 (1993).

    Article  CAS  Google Scholar 

  31. Storgard, C.M. et al. Decreased angiogenesis and arthritic disease in rabbits treated with an αvβ3 antagonist. J. Clin. Invest. 103, 47–54 (1999).

    Article  CAS  Google Scholar 

  32. Jacobson, M.D., Weil, M. & Raff, M.C. Programmed cell death in animal development. Cell 88, 347–354 ( 1997).

    Article  CAS  Google Scholar 

  33. Dragovich, T., Rudin, C.M. & Thompson, C.B. Signal transduction pathways that regulate cell survival and cell death. Oncogene 17, 3207– 3213 (1998).

    Article  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Benjamin, L.E., Golijanin, D., Itin, A., Pode, D. & Keshet, E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J. Clin. Invest. 103, 159–165 (1999).

    Article  CAS  Google Scholar 

  36. Gupta, K. et al. VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling. Exp. Cell Res. 247, 495–504 ( 1999).

    Article  CAS  Google Scholar 

  37. Erpel, T. & Courtneidge, S.A. Src family protein tyrosine kinases and cellular signal transduction pathways. Curr. Opin. Cell Biol. 7, 176–182 ( 1995).

    Article  CAS  Google Scholar 

  38. Miyakawa, T. et al. Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function. Science 278, 698–701 (1997).

    Article  CAS  Google Scholar 

  39. Grant, S.G.N. et al. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science 258, 1903–1910 (1992).

    Article  CAS  Google Scholar 

  40. Juo, P., et al. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases. Mol. Cell. Biol. 17, 24–35 (1997).

    Article  CAS  Google Scholar 

  41. Cardone, M.H., Salvesen, G.S., Widmann, C., Johnson, G. & Frisch, S.M. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell 90, 315–323 (1997).

    Article  CAS  Google Scholar 

  42. Kothakota, S. et al. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278, 294– 298 (1997).

    Article  CAS  Google Scholar 

  43. Salazar, R., Bell, S.E. & Davis, G.E. Coordinate induction of the actin cytoskeletal regulatory proteins gelsolin, vasodilator-stimulated phosphoprotein, and profilin during capillary morphogenesis in vitro. Exp. Cell Res. 249, 22–32 (1999).

    Article  CAS  Google Scholar 

  44. Brooks, P.C. et al. Integrin alphaV beta3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157–1164 (1994).

    Article  CAS  Google Scholar 

  45. Stromblad, S., Becker, J.C., Yebra, M., Brooks, P.C. & Cheresh, D.A. Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis. J. Clin. Invest. 98, 426–433 ( 1996).

    Article  CAS  Google Scholar 

  46. Streilein, J.W. Unraveling immune privilege. Science 270, 1158–1159 (1995).

    Article  CAS  Google Scholar 

  47. Schlaepfer, D.D. & Hunter, T. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol. Cell. Biol. 16, 5623–5633 ( 1996).

    Article  CAS  Google Scholar 

  48. Febbraio, M., Abumrad, N.A., Haijar, D.P., Sharma, K., Pearce, S.F.A. & Silverstein, R.L. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism. J. Biol. Chem. 274, 19055–19062 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Soff (Northwestern University) and M. O'Reilly (Harvard University), for supplying active angiostatin, X. Huang and L. Huang for technical assistance, and the National Cancer Institute for support through grants CA52750 and CA64239. B.J. acknowledges support from a NATO fellowship and also thanks F. Dolfi, J. M. Redondo and P. Gómez del Arco for their advice on kinase assays, D. Dawson for discussions, and A. Munoz for his support.

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Author notes

  1. Benilde Jiménez

    Present address: Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, Arturo Duperier 4, 28029, Madrid, Spain

  2. Benilde Jiménez and Olga V. Volpert: B.J. and O.V.V. contributed equally to this study.

Authors and Affiliations

  1. Department of Microbiology-Immunology and Robert H. Lurie Comprehensive Cancer Center, Chicago, 60611, Illinois, USA

    Benilde Jiménez, Olga V. Volpert, Susan E. Crawford & Noël Bouck

  2. Department of Pathology, Northwestern University Medical School, Chicago, 60611, Illinois, USA

    Susan E. Crawford

  3. Department of Medicine, Division of Hematology-Oncology Cornell University Medical College, New York, 10021, New York, USA

    Maria Febbraio & Roy L. Silverstein

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Jiménez, B., Volpert, O., Crawford, S. et al. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 6, 41–48 (2000). https://doi.org/10.1038/71517

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