Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Sep 15;326(Pt 3):661–668. doi: 10.1042/bj3260661

Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells.

F Bono 1, P Rigon 1, I Lamarche 1, P Savi 1, V Salel 1, J M Herbert 1
PMCID: PMC1218719  PMID: 9307014

Abstract

Basic fibroblast growth factor (bFGF) and its specific receptors have diverse roles on a variety of cell types, such as the induction of vascular smooth-muscle cell proliferation which contributes to restenosis after coronary balloon angioplasty. bFGF is also known to interact with heparan sulphate proteoglycans present on the cell surface or in the extracellular matrix. In this study, the binding of 125I-bFGF to human aortic smooth-muscle cells was investigated. 125I-bFGF binding to these cells was reversible and saturable. Scatchard analysis revealed the presence of two distinct binding sites: a high-affinity receptor (Kd=38+/-7 pM; 1480+/-220 sites/cell) and a low-affinity non-saturable binding site (Kd=8. 0+/-2.0 nM). Pretreatment of the cells with heparinase resulted in a large reduction of 125I-bFGF binding to its low-affinity receptors, suggesting that they are heparin-like molecules. The specificity of the low- and high-affinity binding sites for bFGF was determined with acidic FGF, platelet-derived growth factor-BB and epidermal growth factor, which did not compete for 125I-bFGF binding. Expression of FGF receptor isoforms analysed by reverse transcriptase-PCR revealed the presence of only the type-1 receptor. Binding to low-affinity binding sites was antagonized by heparin, suramin, protamine sulphate and platelet factor 4. Unexpectedly, these molecules also reduced the binding of 125I-bFGF to its high-affinity sites. Consistent with these results, heparin, suramin, protamine sulphate and platelet factor 4 inhibited bFGF-induced proliferation of human aortic smooth-muscle cells. Heparin abrogated bFGF-induced release of tissue-type plasminogen activator by these cells. These observations suggest that the interaction of bFGF with human aortic smooth-muscle cells is different from that described for other cells such as endothelial cells, in which heparin acts as a potentiating factor of the mitogenic activity of bFGF.

Full Text

The Full Text of this article is available as a PDF (526.3 KB).

Selected References

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

  1. Andres J. L., DeFalcis D., Noda M., Massagué J. Binding of two growth factor families to separate domains of the proteoglycan betaglycan. J Biol Chem. 1992 Mar 25;267(9):5927–5930. [PubMed] [Google Scholar]
  2. Baird A., Klagsbrun M. The fibroblast growth factor family. Cancer Cells. 1991 Jun;3(6):239–243. [PubMed] [Google Scholar]
  3. Bashkin P., Doctrow S., Klagsbrun M., Svahn C. M., Folkman J., Vlodavsky I. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry. 1989 Feb 21;28(4):1737–1743. doi: 10.1021/bi00430a047. [DOI] [PubMed] [Google Scholar]
  4. Basilico C., Moscatelli D. The FGF family of growth factors and oncogenes. Adv Cancer Res. 1992;59:115–165. doi: 10.1016/s0065-230x(08)60305-x. [DOI] [PubMed] [Google Scholar]
  5. Bilozur M. E., Biswas C. Identification and characterization of heparan sulfate-binding proteins from human lung carcinoma cells. J Biol Chem. 1990 Nov 15;265(32):19697–19703. [PubMed] [Google Scholar]
  6. Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
  7. Castellot J. J., Jr, Addonizio M. L., Rosenberg R., Karnovsky M. J. Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J Cell Biol. 1981 Aug;90(2):372–379. doi: 10.1083/jcb.90.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castellot J. J., Jr, Cochran D. L., Karnovsky M. J. Effect of heparin on vascular smooth muscle cells. I. Cell metabolism. J Cell Physiol. 1985 Jul;124(1):21–28. doi: 10.1002/jcp.1041240105. [DOI] [PubMed] [Google Scholar]
  9. Castellot J. J., Jr, Wong K., Herman B., Hoover R. L., Albertini D. F., Wright T. C., Caleb B. L., Karnovsky M. J. Binding and internalization of heparin by vascular smooth muscle cells. J Cell Physiol. 1985 Jul;124(1):13–20. doi: 10.1002/jcp.1041240104. [DOI] [PubMed] [Google Scholar]
  10. Clowes A. W., Clowes M. M., Au Y. P., Reidy M. A., Belin D. Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery. Circ Res. 1990 Jul;67(1):61–67. doi: 10.1161/01.res.67.1.61. [DOI] [PubMed] [Google Scholar]
  11. Clowes A. W., Karnowsky M. J. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature. 1977 Feb 17;265(5595):625–626. doi: 10.1038/265625a0. [DOI] [PubMed] [Google Scholar]
  12. Dionne C. A., Crumley G., Bellot F., Kaplow J. M., Searfoss G., Ruta M., Burgess W. H., Jaye M., Schlessinger J. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors. EMBO J. 1990 Sep;9(9):2685–2692. doi: 10.1002/j.1460-2075.1990.tb07454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edelman E. R., Nugent M. A., Smith L. T., Karnovsky M. J. Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries. J Clin Invest. 1992 Feb;89(2):465–473. doi: 10.1172/JCI115607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flaumenhaft R., Moscatelli D., Rifkin D. B. Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor. J Cell Biol. 1990 Oct;111(4):1651–1659. doi: 10.1083/jcb.111.4.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flaumenhaft R., Moscatelli D., Saksela O., Rifkin D. B. Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J Cell Physiol. 1989 Jul;140(1):75–81. doi: 10.1002/jcp.1041400110. [DOI] [PubMed] [Google Scholar]
  16. Fritze L. M., Reilly C. F., Rosenberg R. D. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol. 1985 Apr;100(4):1041–1049. doi: 10.1083/jcb.100.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gallagher J. T. Heparan sulphates as membrane receptors for the fibroblast growth factors. Eur J Clin Chem Clin Biochem. 1994 Apr;32(4):239–247. [PubMed] [Google Scholar]
  18. Gospodarowicz D., Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol. 1986 Sep;128(3):475–484. doi: 10.1002/jcp.1041280317. [DOI] [PubMed] [Google Scholar]
  19. Hoffman R., Paper D. H., Donaldson J., Alban S., Franz G. Characterisation of a laminarin sulphate which inhibits basic fibroblast growth factor binding and endothelial cell proliferation. J Cell Sci. 1995 Nov;108(Pt 11):3591–3598. doi: 10.1242/jcs.108.11.3591. [DOI] [PubMed] [Google Scholar]
  20. Huang S. S., Huang J. S. Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity. J Biol Chem. 1986 Jul 25;261(21):9568–9571. [PubMed] [Google Scholar]
  21. Hughes S. E., Hall P. A. Overview of the fibroblast growth factor and receptor families: complexity, functional diversity, and implications for future cardiovascular research. Cardiovasc Res. 1993 Jul;27(7):1199–1203. doi: 10.1093/cvr/27.7.1199. [DOI] [PubMed] [Google Scholar]
  22. Jacobs S., Chang K-J, Cuatrecasas P. Estimation of hormone receptor affinity by competitive displacement of labeled ligand: effect of concentration of receptor and of labeled ligand. Biochem Biophys Res Commun. 1975 Sep 16;66(2):687–692. doi: 10.1016/0006-291x(75)90564-1. [DOI] [PubMed] [Google Scholar]
  23. Jaye M., Schlessinger J., Dionne C. A. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992 Jun 10;1135(2):185–199. doi: 10.1016/0167-4889(92)90136-y. [DOI] [PubMed] [Google Scholar]
  24. Kan M., Wang F., Xu J., Crabb J. W., Hou J., McKeehan W. L. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science. 1993 Mar 26;259(5103):1918–1921. doi: 10.1126/science.8456318. [DOI] [PubMed] [Google Scholar]
  25. Keegan K., Johnson D. E., Williams L. T., Hayman M. J. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1095–1099. doi: 10.1073/pnas.88.4.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kiefer M. C., Stephans J. C., Crawford K., Okino K., Barr P. J. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6985–6989. doi: 10.1073/pnas.87.18.6985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klagsbrun M., Baird A. A dual receptor system is required for basic fibroblast growth factor activity. Cell. 1991 Oct 18;67(2):229–231. doi: 10.1016/0092-8674(91)90173-v. [DOI] [PubMed] [Google Scholar]
  28. Klagsbrun M. The fibroblast growth factor family: structural and biological properties. Prog Growth Factor Res. 1989;1(4):207–235. doi: 10.1016/0955-2235(89)90012-4. [DOI] [PubMed] [Google Scholar]
  29. Lee P. L., Johnson D. E., Cousens L. S., Fried V. A., Williams L. T. Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science. 1989 Jul 7;245(4913):57–60. doi: 10.1126/science.2544996. [DOI] [PubMed] [Google Scholar]
  30. Lindner V., Olson N. E., Clowes A. W., Reidy M. A. Inhibition of smooth muscle cell proliferation in injured rat arteries. Interaction of heparin with basic fibroblast growth factor. J Clin Invest. 1992 Nov;90(5):2044–2049. doi: 10.1172/JCI116085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lindner V., Reidy M. A. Expression of basic fibroblast growth factor and its receptor by smooth muscle cells and endothelium in injured rat arteries. An en face study. Circ Res. 1993 Sep;73(3):589–595. doi: 10.1161/01.res.73.3.589. [DOI] [PubMed] [Google Scholar]
  32. Moscatelli D. High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J Cell Physiol. 1987 Apr;131(1):123–130. doi: 10.1002/jcp.1041310118. [DOI] [PubMed] [Google Scholar]
  33. Moukadiri H., Gouzi L., Detolle-Sarbach S., Guez D., Plouët J. Inhibition of basic fibroblast growth factor and vasculotropin biological activities on cultured cells by almitrine. Biochim Biophys Acta. 1995 Mar 16;1265(2-3):168–172. doi: 10.1016/0167-4889(94)00215-z. [DOI] [PubMed] [Google Scholar]
  34. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  35. Nugent M. A., Karnovsky M. J., Edelman E. R. Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells. Circ Res. 1993 Dec;73(6):1051–1060. doi: 10.1161/01.res.73.6.1051. [DOI] [PubMed] [Google Scholar]
  36. Olwin B. B., Hauschka S. D. Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts. Biochemistry. 1986 Jun 17;25(12):3487–3492. doi: 10.1021/bi00360a001. [DOI] [PubMed] [Google Scholar]
  37. Partanen J., Mäkelä T. P., Eerola E., Korhonen J., Hirvonen H., Claesson-Welsh L., Alitalo K. FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. EMBO J. 1991 Jun;10(6):1347–1354. doi: 10.1002/j.1460-2075.1991.tb07654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Presta M., Maier J. A., Rusnati M., Ragnotti G. Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form. J Cell Physiol. 1989 Jul;140(1):68–74. doi: 10.1002/jcp.1041400109. [DOI] [PubMed] [Google Scholar]
  39. Rapraeger A. C., Krufka A., Olwin B. B. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science. 1991 Jun 21;252(5013):1705–1708. doi: 10.1126/science.1646484. [DOI] [PubMed] [Google Scholar]
  40. Rogelj S., Klagsbrun M., Atzmon R., Kurokawa M., Haimovitz A., Fuks Z., Vlodavsky I. Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells. J Cell Biol. 1989 Aug;109(2):823–831. doi: 10.1083/jcb.109.2.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roghani M., Mansukhani A., Dell'Era P., Bellosta P., Basilico C., Rifkin D. B., Moscatelli D. Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding. J Biol Chem. 1994 Feb 11;269(6):3976–3984. [PubMed] [Google Scholar]
  42. Roghani M., Moscatelli D. Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms. J Biol Chem. 1992 Nov 5;267(31):22156–22162. [PubMed] [Google Scholar]
  43. Rusnati M., Urbinati C., Presta M. Internalization of basic fibroblast growth factor (bFGF) in cultured endothelial cells: role of the low affinity heparin-like bFGF receptors. J Cell Physiol. 1993 Jan;154(1):152–161. doi: 10.1002/jcp.1041540119. [DOI] [PubMed] [Google Scholar]
  44. Saksela O., Moscatelli D., Sommer A., Rifkin D. B. Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol. 1988 Aug;107(2):743–751. doi: 10.1083/jcb.107.2.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sommer A., Rifkin D. B. Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan. J Cell Physiol. 1989 Jan;138(1):215–220. doi: 10.1002/jcp.1041380129. [DOI] [PubMed] [Google Scholar]
  46. Takano S., Gately S., Neville M. E., Herblin W. F., Gross J. L., Engelhard H., Perricone M., Eidsvoog K., Brem S. Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator. Cancer Res. 1994 May 15;54(10):2654–2660. [PubMed] [Google Scholar]
  47. Vigny M., Ollier-Hartmann M. P., Lavigne M., Fayein N., Jeanny J. C., Laurent M., Courtois Y. Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor. J Cell Physiol. 1988 Nov;137(2):321–328. doi: 10.1002/jcp.1041370216. [DOI] [PubMed] [Google Scholar]
  48. Watson J. B., Getzler S. B., Mosher D. F. Platelet factor 4 modulates the mitogenic activity of basic fibroblast growth factor. J Clin Invest. 1994 Jul;94(1):261–268. doi: 10.1172/JCI117316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xin X., Johnson A. D., Scott-Burden T., Engler D., Casscells W. The predominant form of fibroblast growth factor receptor expressed by proliferating human arterial smooth muscle cells in culture is type I. Biochem Biophys Res Commun. 1994 Oct 28;204(2):557–564. doi: 10.1006/bbrc.1994.2495. [DOI] [PubMed] [Google Scholar]
  50. Yayon A., Klagsbrun M., Esko J. D., Leder P., Ornitz D. M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 Feb 22;64(4):841–848. doi: 10.1016/0092-8674(91)90512-w. [DOI] [PubMed] [Google Scholar]
  51. van Neck J. W., Medina J. J., Onnekink C., Schwartz S. M., Bloemers H. P. Expression of basic fibroblast growth factor and fibroblast growth factor receptor genes in cultured rat aortic smooth muscle cells. Biochim Biophys Acta. 1995 Apr 4;1261(2):210–214. doi: 10.1016/0167-4781(94)00247-z. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES