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. 1997 Dec;151(6):1775–1784.

Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms.

A Karsan 1, E Yee 1, G G Poirier 1, P Zhou 1, R Craig 1, J M Harlan 1
PMCID: PMC1858363  PMID: 9403728

Abstract

Intact endothelium acts as a sensor and transducer of signals and also provides a nonthrombogenic surface at the blood-vascular wall interface. Hence, mechanisms that maintain the integrity of the endothelium are of interest in physiological and pathological states. In this study we show that apoptosis induced by growth factor and serum deprivation of endothelial cells occurs at all phases of the cell cycle and can be blocked by fibroblast growth factor-2 (FGF-2) independently of its mitogenic activity. As the Bcl-2 family of proteins plays a prominent role in regulating cell survival, we attempted to identify Bcl-2 homologues expressed in endothelial cells. Here we demonstrate that, in addition to the previously identified A1, four other members of the Bcl-2 family, Bcl-2, Mcl-1, Bcl-X(L), and Bax, are expressed in endothelial cells. Of these family members, only Bcl-2 is induced by FGF-2. Overexpression of Bcl-2, using a retroviral vector, protects endothelial cells from serum and growth factor deprivation. There is no difference in FGF-2-induced proliferation between Bcl-2-overexpressing cells and those transduced with the empty retroviral vector. At early time points Bcl-2 is not up-regulated, but FGF-2 still has a protective effect. However, FGF-2 protects only adherent endothelial cells but not those that are cultured in suspension. The early effect of FGF-2 is dependent on tyrosine phosphorylation but not on activation of the MAP kinase pathway. Thus, FGF-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki S., Shimada Y., Kaji K., Hayashi H. Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation. Biochem Biophys Res Commun. 1990 May 16;168(3):1194–1200. doi: 10.1016/0006-291x(90)91155-l. [DOI] [PubMed] [Google Scholar]
  2. Boise L. H., González-García M., Postema C. E., Ding L., Lindsten T., Turka L. A., Mao X., Nuñez G., Thompson C. B. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993 Aug 27;74(4):597–608. doi: 10.1016/0092-8674(93)90508-n. [DOI] [PubMed] [Google Scholar]
  3. Bombeli T., Karsan A., Tait J. F., Harlan J. M. Apoptotic vascular endothelial cells become procoagulant. Blood. 1997 Apr 1;89(7):2429–2442. [PubMed] [Google Scholar]
  4. Borner C., Martinou I., Mattmann C., Irmler M., Schaerer E., Martinou J. C., Tschopp J. The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J Cell Biol. 1994 Aug;126(4):1059–1068. doi: 10.1083/jcb.126.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casciola-Rosen L. A., Miller D. K., Anhalt G. J., Rosen A. Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J Biol Chem. 1994 Dec 9;269(49):30757–30760. [PubMed] [Google Scholar]
  6. Cho A., Courtman D. W., Langille B. L. Apoptosis (programmed cell death) in arteries of the neonatal lamb. Circ Res. 1995 Feb;76(2):168–175. doi: 10.1161/01.res.76.2.168. [DOI] [PubMed] [Google Scholar]
  7. Desnoyers S., Shah G. M., Brochu G., Poirier G. G. Erasable blot of poly(ADP-ribose) polymerase. Anal Biochem. 1994 May 1;218(2):470–473. doi: 10.1006/abio.1994.1212. [DOI] [PubMed] [Google Scholar]
  8. Enenstein J., Waleh N. S., Kramer R. H. Basic FGF and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells. Exp Cell Res. 1992 Dec;203(2):499–503. doi: 10.1016/0014-4827(92)90028-7. [DOI] [PubMed] [Google Scholar]
  9. Friesel R. E., Maciag T. Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. FASEB J. 1995 Jul;9(10):919–925. doi: 10.1096/fasebj.9.10.7542215. [DOI] [PubMed] [Google Scholar]
  10. Fuks Z., Persaud R. S., Alfieri A., McLoughlin M., Ehleiter D., Schwartz J. L., Seddon A. P., Cordon-Cardo C., Haimovitz-Friedman A. Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo. Cancer Res. 1994 May 15;54(10):2582–2590. [PubMed] [Google Scholar]
  11. Gardner A. M., Johnson G. L. Fibroblast growth factor-2 suppression of tumor necrosis factor alpha-mediated apoptosis requires Ras and the activation of mitogen-activated protein kinase. J Biol Chem. 1996 Jun 14;271(24):14560–14566. doi: 10.1074/jbc.271.24.14560. [DOI] [PubMed] [Google Scholar]
  12. Garfinkel S., Hu X., Prudovsky I. A., McMahon G. A., Kapnik E. M., McDowell S. D., Maciag T. FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates. J Cell Biol. 1996 Aug;134(3):783–791. doi: 10.1083/jcb.134.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geng Y. J., Libby P. Evidence for apoptosis in advanced human atheroma. Colocalization with interleukin-1 beta-converting enzyme. Am J Pathol. 1995 Aug;147(2):251–266. [PMC free article] [PubMed] [Google Scholar]
  14. Gibbons G. H., Dzau V. J. The emerging concept of vascular remodeling. N Engl J Med. 1994 May 19;330(20):1431–1438. doi: 10.1056/NEJM199405193302008. [DOI] [PubMed] [Google Scholar]
  15. Gjertsen B. T., Døskeland S. O. Protein phosphorylation in apoptosis. Biochim Biophys Acta. 1995 Nov 9;1269(2):187–199. doi: 10.1016/0167-4889(95)00117-b. [DOI] [PubMed] [Google Scholar]
  16. Haldar S., Jena N., Croce C. M. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4507–4511. doi: 10.1073/pnas.92.10.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Han D. K., Haudenschild C. C., Hong M. K., Tinkle B. T., Leon M. B., Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995 Aug;147(2):267–277. [PMC free article] [PubMed] [Google Scholar]
  18. Hobson B., Denekamp J. Endothelial proliferation in tumours and normal tissues: continuous labelling studies. Br J Cancer. 1984 Apr;49(4):405–413. doi: 10.1038/bjc.1984.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  20. Hoyt D. G., Mannix R. J., Rusnak J. M., Pitt B. R., Lazo J. S. Collagen is a survival factor against LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells. Am J Physiol. 1995 Aug;269(2 Pt 1):L171–L177. doi: 10.1152/ajplung.1995.269.2.L171. [DOI] [PubMed] [Google Scholar]
  21. Isner J. M., Kearney M., Bortman S., Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995 Jun 1;91(11):2703–2711. doi: 10.1161/01.cir.91.11.2703. [DOI] [PubMed] [Google Scholar]
  22. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Karsan A., Yee E., Harlan J. M. Endothelial cell death induced by tumor necrosis factor-alpha is inhibited by the Bcl-2 family member, A1. J Biol Chem. 1996 Nov 1;271(44):27201–27204. doi: 10.1074/jbc.271.44.27201. [DOI] [PubMed] [Google Scholar]
  24. Karsan A., Yee E., Kaushansky K., Harlan J. M. Cloning of human Bcl-2 homologue: inflammatory cytokines induce human A1 in cultured endothelial cells. Blood. 1996 Apr 15;87(8):3089–3096. [PubMed] [Google Scholar]
  25. Kaufmann S. H., Desnoyers S., Ottaviano Y., Davidson N. E., Poirier G. G. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993 Sep 1;53(17):3976–3985. [PubMed] [Google Scholar]
  26. Kinoshita T., Yokota T., Arai K., Miyajima A. Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors. EMBO J. 1995 Jan 16;14(2):266–275. doi: 10.1002/j.1460-2075.1995.tb07000.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klein S., Giancotti F. G., Presta M., Albelda S. M., Buck C. A., Rifkin D. B. Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells. Mol Biol Cell. 1993 Oct;4(10):973–982. doi: 10.1091/mbc.4.10.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knudson C. M., Tung K. S., Tourtellotte W. G., Brown G. A., Korsmeyer S. J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 1995 Oct 6;270(5233):96–99. doi: 10.1126/science.270.5233.96. [DOI] [PubMed] [Google Scholar]
  29. Kondo S., Yin D., Aoki T., Takahashi J. A., Morimura T., Takeuchi J. bcl-2 gene prevents apoptosis of basic fibroblast growth factor-deprived murine aortic endothelial cells. Exp Cell Res. 1994 Aug;213(2):428–432. doi: 10.1006/excr.1994.1219. [DOI] [PubMed] [Google Scholar]
  30. Kozopas K. M., Yang T., Buchan H. L., Zhou P., Craig R. W. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3516–3520. doi: 10.1073/pnas.90.8.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamarre D., Talbot B., de Murcia G., Laplante C., Leduc Y., Mazen A., Poirier G. G. Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study. Biochim Biophys Acta. 1988 Jul 13;950(2):147–160. doi: 10.1016/0167-4781(88)90007-3. [DOI] [PubMed] [Google Scholar]
  32. Lin E. Y., Orlofsky A., Wang H. G., Reed J. C., Prystowsky M. B. A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation. Blood. 1996 Feb 1;87(3):983–992. [PubMed] [Google Scholar]
  33. Maciag T., Cerundolo J., Ilsley S., Kelley P. R., Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5674–5678. doi: 10.1073/pnas.76.11.5674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Martin S. J., O'Brien G. A., Nishioka W. K., McGahon A. J., Mahboubi A., Saido T. C., Green D. R. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem. 1995 Mar 24;270(12):6425–6428. doi: 10.1074/jbc.270.12.6425. [DOI] [PubMed] [Google Scholar]
  35. Meredith J. E., Jr, Fazeli B., Schwartz M. A. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993 Sep;4(9):953–961. doi: 10.1091/mbc.4.9.953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller A. D., Miller D. G., Garcia J. V., Lynch C. M. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 1993;217:581–599. doi: 10.1016/0076-6879(93)17090-r. [DOI] [PubMed] [Google Scholar]
  37. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  38. Nuñez G., London L., Hockenbery D., Alexander M., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed] [Google Scholar]
  39. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  40. Pearson J. D. Endothelial cell function and thrombosis. Baillieres Clin Haematol. 1994 Sep;7(3):441–452. doi: 10.1016/s0950-3536(05)80092-7. [DOI] [PubMed] [Google Scholar]
  41. Pober J. S., Cotran R. S. Cytokines and endothelial cell biology. Physiol Rev. 1990 Apr;70(2):427–451. doi: 10.1152/physrev.1990.70.2.427. [DOI] [PubMed] [Google Scholar]
  42. Re F., Zanetti A., Sironi M., Polentarutti N., Lanfrancone L., Dejana E., Colotta F. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol. 1994 Oct;127(2):537–546. doi: 10.1083/jcb.127.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reed J. C. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994 Jan;124(1-2):1–6. doi: 10.1083/jcb.124.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Reynolds J. E., Yang T., Qian L., Jenkinson J. D., Zhou P., Eastman A., Craig R. W. Mcl-1, a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells. Cancer Res. 1994 Dec 15;54(24):6348–6352. [PubMed] [Google Scholar]
  45. Ruoslahti E., Reed J. C. Anchorage dependence, integrins, and apoptosis. Cell. 1994 May 20;77(4):477–478. doi: 10.1016/0092-8674(94)90209-7. [DOI] [PubMed] [Google Scholar]
  46. Sato T., Hanada M., Bodrug S., Irie S., Iwama N., Boise L. H., Thompson C. B., Golemis E., Fong L., Wang H. G. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9238–9242. doi: 10.1073/pnas.91.20.9238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sedlak T. W., Oltvai Z. N., Yang E., Wang K., Boise L. H., Thompson C. B., Korsmeyer S. J. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7834–7838. doi: 10.1073/pnas.92.17.7834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sentman C. L., Shutter J. R., Hockenbery D., Kanagawa O., Korsmeyer S. J. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell. 1991 Nov 29;67(5):879–888. doi: 10.1016/0092-8674(91)90361-2. [DOI] [PubMed] [Google Scholar]
  49. Strömblad 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. 1996 Jul 15;98(2):426–433. doi: 10.1172/JCI118808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Varani J., Dame M. K., Taylor C. G., Sarma V., Merino R., Kunkel R. G., Nunez G., Dixit V. M. Age-dependent injury in human umbilical vein endothelial cells: relationship to apoptosis and correlation with a lack of A20 expression. Lab Invest. 1995 Dec;73(6):851–858. [PubMed] [Google Scholar]
  51. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  52. Wang E. Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res. 1995 Jun 1;55(11):2284–2292. [PubMed] [Google Scholar]
  53. Wary K. K., Mainiero F., Isakoff S. J., Marcantonio E. E., Giancotti F. G. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell. 1996 Nov 15;87(4):733–743. doi: 10.1016/s0092-8674(00)81392-6. [DOI] [PubMed] [Google Scholar]
  54. Yang T., Kozopas K. M., Craig R. W. The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2. J Cell Biol. 1995 Mar;128(6):1173–1184. doi: 10.1083/jcb.128.6.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yin X. M., Oltvai Z. N., Korsmeyer S. J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature. 1994 May 26;369(6478):321–323. doi: 10.1038/369321a0. [DOI] [PubMed] [Google Scholar]

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