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. 1988 Aug 1;107(2):743–751. doi: 10.1083/jcb.107.2.743

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation

PMCID: PMC2115214  PMID: 2971068

Abstract

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

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Selected References

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  1. Akama T., Yamada K. M., Seno N., Matsumoto I., Kono I., Kashiwagi H., Funaki T., Hayashi M. Immunological characterization of human vitronectin and its binding to glycosaminoglycans. J Biochem. 1986 Nov;100(5):1343–1351. doi: 10.1093/oxfordjournals.jbchem.a121840. [DOI] [PubMed] [Google Scholar]
  2. Baird A., Ling N. Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun. 1987 Jan 30;142(2):428–435. doi: 10.1016/0006-291x(87)90292-0. [DOI] [PubMed] [Google Scholar]
  3. Carey D. J., Rafferty C. M., Schramm M. M. Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver. J Biol Chem. 1987 Mar 5;262(7):3376–3381. [PubMed] [Google Scholar]
  4. 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]
  5. Cole G. J., Schubert D., Glaser L. Cell-substratum adhesion in chick neural retina depends upon protein-heparan sulfate interactions. J Cell Biol. 1985 Apr;100(4):1192–1199. doi: 10.1083/jcb.100.4.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Folkman J., Haudenschild C. C., Zetter B. R. Long-term culture of capillary endothelial cells. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5217–5221. doi: 10.1073/pnas.76.10.5217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Gospodarowicz D., Ferrara N., Schweigerer L., Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev. 1987 May;8(2):95–114. doi: 10.1210/edrv-8-2-95. [DOI] [PubMed] [Google Scholar]
  10. Gross J. L., Moscatelli D., Jaffe E. A., Rifkin D. B. Plasminogen activator and collagenase production by cultured capillary endothelial cells. J Cell Biol. 1982 Dec;95(3):974–981. doi: 10.1083/jcb.95.3.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hascall V. C., Kimura J. H. Proteoglycans: isolation and characterization. Methods Enzymol. 1982;82(Pt A):769–800. doi: 10.1016/0076-6879(82)82102-2. [DOI] [PubMed] [Google Scholar]
  12. Hassell J. R., Kimura J. H., Hascall V. C. Proteoglycan core protein families. Annu Rev Biochem. 1986;55:539–567. doi: 10.1146/annurev.bi.55.070186.002543. [DOI] [PubMed] [Google Scholar]
  13. Hök M., Kjellén L., Johansson S. Cell-surface glycosaminoglycans. Annu Rev Biochem. 1984;53:847–869. doi: 10.1146/annurev.bi.53.070184.004215. [DOI] [PubMed] [Google Scholar]
  14. Iozzo R. V. Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface. J Cell Biol. 1984 Aug;99(2):403–417. doi: 10.1083/jcb.99.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iozzo R. V. Turnover of heparan sulfate proteoglycan in human colon carcinoma cells. A quantitative biochemical and autoradiographic study. J Biol Chem. 1987 Feb 5;262(4):1888–1900. [PubMed] [Google Scholar]
  16. Isemura M., Sato N., Yamaguchi Y., Aikawa J., Munakata H., Hayashi N., Yosizawa Z., Nakamura T., Kubota A., Arakawa M. Isolation and characterization of fibronectin-binding proteoglycan carrying both heparan sulfate and dermatan sulfate chains from human placenta. J Biol Chem. 1987 Jun 25;262(18):8926–8933. [PubMed] [Google Scholar]
  17. Kjellén L., Pettersson I., Hök M. Cell-surface heparan sulfate: an intercalated membrane proteoglycan. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5371–5375. doi: 10.1073/pnas.78.9.5371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Klagsbrun M., Sasse J., Sullivan R., Smith J. A. Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2448–2452. doi: 10.1073/pnas.83.8.2448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kramer R. H., Vogel K. G., Nicolson G. L. Solubilization and degradation of subendothelial matrix glycoproteins and proteoglycans by metastatic tumor cells. J Biol Chem. 1982 Mar 10;257(5):2678–2686. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Laterra J., Silbert J. E., Culp L. A. Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin. J Cell Biol. 1983 Jan;96(1):112–123. doi: 10.1083/jcb.96.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lobb R. R., Fett J. W. Purification of two distinct growth factors from bovine neural tissue by heparin affinity chromatography. Biochemistry. 1984 Dec 18;23(26):6295–6299. doi: 10.1021/bi00321a001. [DOI] [PubMed] [Google Scholar]
  23. Marcum J. A., Atha D. H., Fritze L. M., Nawroth P., Stern D., Rosenberg R. D. Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J Biol Chem. 1986 Jun 5;261(16):7507–7517. [PubMed] [Google Scholar]
  24. Moscatelli D., Presta M., Joseph-Silverstein J., Rifkin D. B. Both normal and tumor cells produce basic fibroblast growth factor. J Cell Physiol. 1986 Nov;129(2):273–276. doi: 10.1002/jcp.1041290220. [DOI] [PubMed] [Google Scholar]
  25. Moscatelli D., Presta M., Rifkin D. B. Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2091–2095. doi: 10.1073/pnas.83.7.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nader H. B., Dietrich C. P., Buonassisi V., Colburn P. Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3565–3569. doi: 10.1073/pnas.84.11.3565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Neufeld G., Gospodarowicz D. The identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells. J Biol Chem. 1985 Nov 5;260(25):13860–13868. [PubMed] [Google Scholar]
  28. 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]
  29. Presta M., Moscatelli D., Joseph-Silverstein J., Rifkin D. B. Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration. Mol Cell Biol. 1986 Nov;6(11):4060–4066. doi: 10.1128/mcb.6.11.4060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reilly C. F., Fritze L. M., Rosenberg R. D. Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J Cell Physiol. 1987 May;131(2):149–157. doi: 10.1002/jcp.1041310203. [DOI] [PubMed] [Google Scholar]
  31. Sommer A., Brewer M. T., Thompson R. C., Moscatelli D., Presta M., Rifkin D. B. A form of human basic fibroblast growth factor with an extended amino terminus. Biochem Biophys Res Commun. 1987 Apr 29;144(2):543–550. doi: 10.1016/s0006-291x(87)80001-3. [DOI] [PubMed] [Google Scholar]
  32. Stamatoglou S. C., Keller J. M. Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen. J Cell Biol. 1983 Jun;96(6):1820–1823. doi: 10.1083/jcb.96.6.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vlodavsky I., Folkman J., Sullivan R., Fridman R., Ishai-Michaeli R., Sasse J., Klagsbrun M. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2292–2296. doi: 10.1073/pnas.84.8.2292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wright T. C., Jr, Johnstone T. V., Castellot J. J., Karnovsky M. J. Inhibition of rat cervical epithelial cell growth by heparin and its reversal by EGF. J Cell Physiol. 1985 Dec;125(3):499–506. doi: 10.1002/jcp.1041250320. [DOI] [PubMed] [Google Scholar]

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