FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023552163> ?p ?o ?g. }

• W2023552163 endingPage "4886" @default.
• W2023552163 startingPage "4879" @default.
• W2023552163 abstract "The growth promoting activity of the subendothelial extracellular matrix (ECM) is attributed to sequestration of basic fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The present study focuses on the growth promoting activity and bFGF binding capacity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintained in the presence of chlorate, an inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM containing 10-15% of the sulfate normally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of 125I-bFGF to sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to release by heparin. Incubation of 125I-bFGF on top of native ECM resulted in dimerization of the ECM-bound bFGF, but there was a markedly reduced binding and dimerization of bFGF on sulfate-depleted ECM. ECM produced in the presence of chlorate contained a nearly 10-fold less endogenous bFGF as compared to native ECM and exerted little or no mitogenic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulated the proliferation of chlorate-treated EC seeded on native ECM, suggesting its interaction with ECM-bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin had no effect on chlorate-treated cells seeded in contact with sulfate-depleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated with the cell surface and ECM act in concert to regulate the bioavailability and growth promoting activity of bFGF. While HS in the subendothelial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to high affinity signal transducing receptors. The growth promoting activity of the subendothelial extracellular matrix (ECM) is attributed to sequestration of basic fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The present study focuses on the growth promoting activity and bFGF binding capacity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintained in the presence of chlorate, an inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM containing 10-15% of the sulfate normally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of 125I-bFGF to sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to release by heparin. Incubation of 125I-bFGF on top of native ECM resulted in dimerization of the ECM-bound bFGF, but there was a markedly reduced binding and dimerization of bFGF on sulfate-depleted ECM. ECM produced in the presence of chlorate contained a nearly 10-fold less endogenous bFGF as compared to native ECM and exerted little or no mitogenic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulated the proliferation of chlorate-treated EC seeded on native ECM, suggesting its interaction with ECM-bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin had no effect on chlorate-treated cells seeded in contact with sulfate-depleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated with the cell surface and ECM act in concert to regulate the bioavailability and growth promoting activity of bFGF. While HS in the subendothelial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to high affinity signal transducing receptors. Heparan sulfate (HS) ( 1The abbreviations used are: HSheparan sulfatebFGFbasic fibroblast growth factorDMEMDulbecco's modified Eagle's mediumECendothelial cellsECMextracellular matrixHSPGheparan sulfate proteoglycansPBSphosphate-buffered salineDSSdisuccinimidyl suberatePAGEpolyacrylamide gel electrophoresis.) is a most ubiquitous glycosaminoglycan present on cell surfaces, in basement membranes and extracellular matrices (Gallagher et al., 10.Gallagher J.T. Lyon M. Steward W.P. Biochem. J. 1986; 236: 313-325Crossref PubMed Scopus (361) Google Scholar; Jackson et al., 1991; Kjellen and Lindahl, 26.Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1675) Google Scholar). Recent interest in heparan sulfate proteoglycans (HSPG) stems from increasing awareness of the functional implications of their interactions with growth factors, matrix molecules, and cytoskeletal elements (Gitay-Goren et al., 11.Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar; Jackson et al., 1991; Ruoslahti and Yamaguchi, 35.Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Abstract Full Text PDF PubMed Scopus (1167) Google Scholar; Vlodavsky et al., 44.Vlodavsky I. Bar-Shavit R. Korner G. Fuks Z. Timpl D.H.R. Orlando R. Basement Membranes: Cellular and Molecular Aspects. Academic Press Inc., Orlando, FL1993: 327-343Google Scholar; Yayon et al., 45.Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2083) Google Scholar). The HS chains have been implicated in a variety of physiological processes including the regulation of glomerular basement membrane permeability to proteins, assembly of basement membranes, regulation of nuclear metabolism, cell attachment and spreading, recruitment of inflammatory cells (chemokines), and the regulation of mammalian cell proliferation and differentiation (Gallagher et al., 1986; Jackson et al., 1991; Ruoslahti and Yamaguchi, 1991; Tanaka et al., 38.Tanaka Y. Adams D.H. Shaw S. Immunol. Today. 1993; 14: 111-115Abstract Full Text PDF PubMed Scopus (382) Google Scholar). The sulfate residues, which may be present on four different positions of the polysaccharide backbone, are of high interest, since they have been shown to be major factors in the determination of specificity in protein-polysaccharide interactions (Lindahl, 27.Lindahl U. Lane D.A. Lindahl U. Heparin: Chemical and Biological Properties, Clinical Applications. Edward Arnold, London1989: 159-189Google Scholar). Of particular significance is the interaction between HS and basic fibroblast growth factor (bFGF), involved in bFGF receptor binding and signal transduction (Ornitz et al., 31.Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (560) Google Scholar; Rapraeger et al., 33.Rapraeger A. Krufka A. Olwin B.R. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1291) Google Scholar; Yayon et al., 1991). A unique, highly sulfated bFGF-binding fragment of HS was isolated from cell surface HSPG of fibroblasts (Turnbull et al., 39.Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar). Sulfation in critical positions along the polysaccharide chain, particularly 2-O-sulfation, seems necessary to generate a specific bFGF binding motif that can support high affinity bFGF-receptor binding and activation (Aviezer et al., 2.Aviezer D. Levy E. Safran M. Svahn C. Buddecke E. Schmidt A. David G. Vlodavsky I. Yayon A. J. Biol. Chem. 1994; 269: 114-121Abstract Full Text PDF PubMed Google Scholar; Habuchi et al., 19.Habuchi H. Suzuki S. Saito T. Tamura T. Harada T. Yoshida K. Kimata K. Biochem. J. 1992; 285: 805-813Crossref PubMed Scopus (163) Google Scholar; Ishihara et al., 23.Ishihara M. Tyrell D.J. Stauber G.B. Brown S. Cousens L. Stack R.J. J. Biol. Chem. 1993; 263: 4675-4683Abstract Full Text PDF Google Scholar; Maccarana et al., 28.Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar; Turnbull et al., 1992). heparan sulfate basic fibroblast growth factor Dulbecco's modified Eagle's medium endothelial cells extracellular matrix heparan sulfate proteoglycans phosphate-buffered saline disuccinimidyl suberate polyacrylamide gel electrophoresis. Chlorate, an inhibitor of ATP sulfurylase and hence of the production of phosphoadenosine phosphosulfate, the active sulfate donor for sulfotransferases (Baeuerle and Huttner, 3.Baeuerle P.A. Huttner W.B. Biochem. Biophys. Res. Commu. 1986; 141: 870-877Crossref PubMed Scopus (292) Google Scholar), has been shown to abolish sulfation on proteins and carbohydrate residues in intact cells without inhibiting cell growth or protein synthesis (Baeuerle and Huttner, 1986; Keller et al., 25.Keller K.M. Brauer P.R. Keller J.M. Biochemistry. 1989; 28: 8100-8107Crossref PubMed Scopus (95) Google Scholar). Exposure to chlorate markedly reduced binding of bFGF to high affinity cell surface receptors and the ability of 3T3 fibroblasts to proliferate in response to bFGF (Guimond et al., 18.Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar; Rapraeger et al., 1991). Our studies on the control of cell proliferation by its local environment focus on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal endothelial cells (EC) (Gospodarowicz et al., 16.Gospodarowicz D. Delgado D. Vlodavsky I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4094-4098Crossref PubMed Scopus (333) Google Scholar; Vlodavsky et al., 40.Vlodavsky I. Liu G.M. Gospodarowicz D. Cell. 1980; 19: 607-616Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 41.Vlodavsky I. Fuks Z. Bar-Ner M. Ariav Y. Schirrmacher V. Cancer Res. 1983; 43: 2704-2711PubMed Google Scholar). This ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular organization. It contains collagens (mostly types III and IV, with smaller amounts of types I and V), proteoglycans (mostly HS- and dermatan sulfate-proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin, and elastin. EC and other cell types plated in contact with this ECM no longer require the addition of soluble bFGF in order to proliferate and express their differentiated functions (Gospodarowicz et al., 1980; Vlodavsky et al., 1980). In subsequent studies bFGF was identified as a complex with HSPG in the subendothelial ECM produced in vitro (Bashkin et al., 4.Bashkin P. Doctrow S. Klagsbrun M. Svahn C.M. Folkman J. Vlodavsky I. Biochemistry. 1989; 28: 1737-1743Crossref PubMed Scopus (516) Google Scholar; Vlodavsky et al., 42.Vlodavsky I. Folkman J. Sullivan R. Fridman R. Ishai-Michaeli R. Sasse J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2292-2296Crossref PubMed Scopus (841) Google Scholar) and on cell surfaces and basement membranes of diverse tissues and blood vessels (Cardon-Cardo et al., 7.Cardon-Cardo C. Vlodavsky I. Haimovitz-Friedman A. Hicklin D. Fuks Z. Lab. Invest. 1990; 63: 832-840PubMed Google Scholar; Gonzalez et al., 13.Gonzalez, A. M., Buscaglia, M., Ong, M., Baird, A. (1990) J. Cell Biol. 110, 753-765Google Scholar). HS-bound bFGF is protected against heat inactivation and proteolytic degradation (Saksela et al., 36.Saksela O. Moscatelli D. Sommer A. Rifkin D.B. J. Cell Biol. 1988; 107: 743-751Crossref PubMed Scopus (652) Google Scholar) and can be released in an active form by heparin-like molecules and HS degrading enzymes (Bashkin et al., 1989; Ishai-Michaeli et al., 21.Ishai-Michaeli R. Eldor A. Vlodavsky I. Cell Reg. 1990; 1: 833-842Crossref PubMed Scopus (171) Google Scholar, 22.Ishai-Michaeli R. Svahn C.M. Chajek-Shaul T. Korner G. Ekre H.P. Vlodavsky I. Biochemistry. 1992; 31: 2080-2088Crossref PubMed Scopus (75) Google Scholar; Vlodavsky et al., 43.Vlodavsky I. Bar-Shavit R. Ishai-Michaeli R. Bashkin P. Fuks Z. Trends Biochem. Sci. 1991; 16: 268-271Abstract Full Text PDF PubMed Scopus (254) Google Scholar), or by proteases (Benezra et al., 5.Benezra M. Vlodavsky I. Ishai-Michaeli R. Neufeld G. Bar-Shavit R. Blood. 1993; 81: 3324-3331Crossref PubMed Google Scholar; Saksela et al., 1988). On the basis of these results, the ECM is regarded as a storage depot for bFGF and possibly other heparin-binding growth factors and cytokines. These immobilized growth factors are held responsible for the growth promoting activity of the ECM. In the present study, corneal EC were cultured in the presence of chlorate to produce sulfate-depleted ECM. This ECM was analyzed for its ability to sequester and dimerize bFGF and its growth promoting activity toward vascular EC and 3T3 fibroblasts, in the absence and presence of exogenously added heparin. We have also analyzed the ability of chlorate-treated EC to respond to native and sulfate-depleted ECM. Recombinant human bFGF was kindly provided by Takeda Chemical Industries (Osaka, Japan). Sepharose 6B was from Pharmacia (Uppsala, Sweden). Sodium heparin from porcine intestinal mucosa (PM-heparin, Mr 14,000, anti-FXa 165 IU/mg) was obtained from Hepar Industries (Franklin, OH). Dulbecco's modified Eagle's medium (DMEM, 1 g of glucose/liter or 4.5 g glucose/liter), Fisher medium (sulfate-free), fetal calf serum, calf serum, penicillin, and streptomycin were obtained from Life Technologies, Inc. Saline containing 0.05% trypsin, 0.01 M sodium phosphate, and 0.02% EDTA (STV) was obtained from Biological Industries (Beit-Haemek, Israel). Tissue culture dishes and multiwell plates were obtained from Nunc (Roskilde, Denmark). [3H]Thymidine, Na235SO4 and Na125I were obtained from Amersham (Buckinghamshire, United Kingdom). Disuccinimidyl suberate (DSS) was purchased from Pierce, and sodium chlorate was purchased from Aldrich. Triton X-100, dextran T-40, and all other chemicals were of reagent grade, purchased from Sigma. Balb/c 3T3 cells were maintained in DMEM (4.5 g of glucose/liter) supplemented with 10% fetal calf serum, penicillin (50 units/ml), and streptomycin (50 μg/ml) at 37°C in a 10% CO2 humidified incubator. Cultures of bovine corneal EC were established from steer eyes as described previously (Bashkin et al., 1989; Gospodarowicz et al., 15.Gospodarowicz D. Mescher A.L. Birdwell C.R. Exp. Eye Res. 1977; 25: 75-89Crossref PubMed Scopus (264) Google Scholar). Stock cultures were maintained in DMEM (1 g of glucose/liter) supplemented with 10% newborn calf serum, 5% fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37°C in a 10% CO2 humidified incubator. Recombinant human bFGF (1 ng/ml) was added every other day during the phase of active cell growth. Bovine aortic EC were cloned and cultured in DMEM (1 g of glucose/liter) supplemented with 10% calf serum, as described (Gospodarowicz et al., 14.Gospodarowicz D. Moran J. Braun D. Birdwell C.R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4120-4124Crossref PubMed Scopus (350) Google Scholar; Vlodavsky et al., 1987). Bovine corneal EC were dissociated from stock cultures (second to fifth passage) with STV and plated into four-well plates at an initial density of 2 × 105 cells/ml/well. Cells were maintained as described above, except that 5% dextran T-40 was included in the growth medium (Gospodarowicz et al., 1980; Vlodavsky et al., 1980). The cells were cultured in the absence and presence of sodium chlorate (30 mM, except when stated otherwise) without replacing the medium and without addition of bFGF. Ten to 12 days after seeding, the subendothelial ECM was exposed by dissolving (5 min, room temperature) the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH4OH, followed by four washes in PBS. The ECM remained intact, free of cellular debris, and firmly attached to the entire area of the tissue culture dish (Gospodarowicz et al., 1980, 17.Gospodarowicz D. Gonzalez R. Fujii D.K. J. Cell. Physiol. 1983; 1983: 191-201Crossref Scopus (71) Google Scholar; Vlodavsky et al., 1980, 1987). For preparation of sulfate-labeled ECM, corneal EC were plated into four-well plates and cultured as described above. Na235SO4 (540-590 mCi/mmol) was added (20 μCi/ml) 1 and 5 days after seeding, and the cultures were incubated with the label without medium change. Ten to 12 days after seeding, the cell monolayer was dissolved and the ECM exposed, as described above. To determine the total amount of sulfate labeled material, the ECM was digested with trypsin (25 μg/ml, 24 h, 37°C) and the solubilized material counted in a β-counter. Protein was determined in aliquots of the trypsinized material using the Coomassie protein assay reagent (Pierce) according to the manufacturer's instructions. To determine the amount of sulfate labeled HS, the ECM was digested (48 h, 37°C, pH 6.2) with a human placental heparanase (endo-β-D-glucuronidase) purified and characterized as described (Gilat et al., 12.Gilat D. Hershkoviz R. Goldkorn I. Cahalon L. Korner G. Vlodavsky I. Lider O. J. Exp. Med. 1995; 181: 1929-1934Crossref PubMed Scopus (112) Google Scholar). The estimated Mr of the HS fragments was 5,000-7,000 as compared to a Mr of about 30,000 ascribed to intact HS side chains released from ECM by treatment with alkaline borohydride or papain (Vlodavsky et al., 1983). Sulfate-labeled low Mr degradation products released into the incubation medium were analyzed by gel filtration on Sepharose 6B, as described (Ishai-Michaeli et al., 1990). Recombinant bFGF was iodinated using chloramine T, as described (Benezra et al., 1993). The specific activity was 1.2-1.7 × 105 cpm/ng bFGF, and the labeled preparation was kept for up to 6 weeks at −70°C. 125I-bFGF binding to ECM was performed as described (Ishai-Michaeli et al., 1992). Unbound bFGF was removed and the remaining ECM was solubilized with 1 N NaOH and counted in a γ-counter. Displacement of ECM-bound 125I-bFGF by heparin was performed as described (Ishai-Michaeli et al., 1992). bFGF cross-linking experiments were carried out as described (Ornitz et al., 1992; Spivak-Kroizman et al., 37.Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (593) Google Scholar). Briefly, 125I-bFGF (25 ng/0.25 ml/well) was incubated (1 h, 24°C) with ECM (four-well plates) in a buffer containing 150 mM NaCl and 25 mM HEPES (pH 7.5). The incubation medium was removed, the dishes were washed three times with PBS followed by incubation (30 min, 24°C) with 0.15 mM DSS in PBS. The cross-linking reaction was quenched with 10 mM ethanolamine-HCl (pH 8.0) for 30 min, the incubation medium removed, and the remaining ECM scraped and dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (150 μl/well, 1 h, 37°C). The soluble material was analyzed by 15% SDS-PAGE. The cross-linked bFGF was visualized by autoradiography using Kodak XAR film. Heparin was reacted with Bolton-Hunter reagent and iodinated as described (Dawes and Pepper, 9.Dawes J. Pepper D.S. Throm. Res. 1979; 14: 845-860Abstract Full Text PDF PubMed Scopus (95) Google Scholar). The specific activity was 6,000 cpm/ng. Binding of 125I-heparin to ECM was performed in the absence and presence of 20 μg/ml unlabeled heparin. Assay for DNA synthesis in growth-arrested Balb/c 3T3 cells was performed as described (Vlodavsky et al., 1987). For measurements of EC proliferation, cells were seeded on ECM or regular tissue culture plastic at a low density (1 × 103/16-mm well) in 0.5 ml DMEM containing 10% heat-inactivated calf serum. Five to 6 days after seeding, the cells were dissociated with STV and counted in a Coulter counter (Coulter Electronics). Alternatively, the cells were exposed (3-4 h, 37°C) to [3H]thymidine (5 μCi/well) and DNA synthesis was determined by measuring the radioactivity incorporated into trichloroacetic acid-insoluble material (Ishai-Michaeli et al., 1990). Thymidine incorporation was linearly correlated to the number of cells per well. In other experiments, the EC were seeded at a clonal cell density (300 cells/35-mm dish) and cell colonies were fixed and stained with 0.1% crystal violet, 10 days after seeding (Gospodarowicz et al., 1976; Vlodavsky et al., 1987). Corneal EC were seeded at a confluent cell density into four-well plates, in the absence and presence of increasing concentrations of chlorate (0.1-60 mM). Under these conditions, a contact inhibited cell monolayer was formed within 6 h and there was no effect to chlorate on the cell number and morphological appearance, thereafter. The cells were maintained in sulfate-free Fisher medium containing 20 μCi/ml Na235SO4 as described under “Experimental Procedures.” Ten days after seeding, the cell layer was dissolved to expose the subendothelial ECM. The ECM was then subjected to complete digestion with trypsin (25 μg/ml, 24 h, 37°C) and the digest counted in a β-counter. Eighty-90% decrease in sulfate content was obtained in ECM produced in the presence of 30 mM chlorate (Fig. 1, inset). Similar results were obtained when the stock of corneal EC was treated (48 h) with 30 mM chlorate before seeding into the four-well plates and thereafter. At this concentration, chlorate had little or no effect on the total amount of ECM protein deposited by the cells, nor on the morphology and organization of the ECM as revealed by phase and scanning electron microscopy. Exposure to 60 mM chlorate resulted in an almost complete inhibition of sulfate incorporation, but this was associated with a slight decrease (<20%) in ECM deposition. In subsequent experiments, sulfate-depleted ECM was produced by cells maintained in the presence of 30 mM chlorate, as described above. Specific incorporation of sulfate into HS was analyzed by measurements of sulfate-labeled material released from ECM during incubation (24 h, 37°C) with a highly purified preparation of human placental heparanase. Sulfate-labeled HS degradation products released into the incubation medium were analyzed by gel filtration on Sepharose 6B, as described (Ishai-Michaeli et al., 1990). As demonstrated in Fig. 1, there was little (<10%) release of sulfate-labeled HS degradation products (fractions 15-35, 0.5 < Kav < 0.8) from ECM produced in the presence of 30 mM chlorate. Sulfate groups are involved in bFGF binding to isolated heparin and HS (Aviezer et al., 1994b; Habuchi et al., 1992; Ishai-Michaeli et al., 1992, 1993; Maccarana et al., 1993; Turnbull et al., 1992). The availability of sulfate-deficient ECM provided an appropriate means to investigate to what extent sulfate groups are involved in bFGF binding to a multimolecular structure such as intact subendothelial ECM. Binding of 125I-bFGF to ECM produced in the presence of 30 mM chlorate was inhibited by 50-60%. Sulfate depletion had a more pronounced effect on bFGF binding to HS in the ECM, as revealed by 70-80% reduction in the amount of ECM-bound bFGF displaced by heparin (Fig. 2). A similar value was obtained when the ECM-HS was degraded by human placental heparanase (data not shown). While heparin or heparanase treatment released 50-60% of the 125I-bFGF bound to native ECM, only 10-15% of the bFGF bound to sulfate-depleted ECM was released under the same conditions. It is therefore conceivable that binding of bFGF to sulfate-depleted ECM produced in the presence of 30 mM chlorate was due primarily to binding to other components of the ECM (i.e. fibronectin) or to glycosaminoglycan side chains deprived of their sulfate moieties (Ornitz et al., 32.Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). We have previously demonstrated that EC and other cell types plated in contact with the subendothelial ECM no longer require the addition of soluble bFGF in order to proliferate (Gospodarowicz et al., 1980; Vlodavsky et al., 1987). This mitogenic effect was attributed primarily to the presence of bFGF in ECM, although the mode of bFGF deposition was not elucidated (Vlodavsky et al., 1987). ECM produced in the absence and presence of chlorate was tested for mitogenic activity toward vascular EC and 3T3 fibroblasts. For this purpose, vascular EC were seeded at a low cell density (1,000 cells/16-mm well) on top of ECM produced in the absence and presence of 30 mM chlorate. Six days after seeding, the cultures were exposed to [3H]thymidine and the amount of trichloroacetic acid-precipitable radioactivity was determined 3 h afterwards (Fig. 3A). EC were also seeded at a clonal cell density (300 cells/35-mm dish) on top of native and sulfate-depleted ECM and cell colonies were stained 10 days after seeding (Fig. 3, inset). As demonstrated in Fig. 3A, ECM produced in the presence of chlorate exerted a greatly reduced mitogenic activity toward vascular EC seeded at a low or clonal cell density. In other experiments (Fig. 3B), ECM produced in the presence of increasing concentrations of chlorate was subjected to trypsin digestion and aliquots of the solubilized material were added to confluent, growth-arrested 3T3 fibroblasts (Fig. 3B) or to sparsely seeded EC (1,000 cells/16-mm well) maintained in the presence of 10% heat-inactivated calf serum (data not shown). A trypsin digest of native ECM was highly mitogenic to growth arrested 3T3 fibroblasts (Fig. 3B), and this activity was inhibited by neutralizing anti-bFGF antibodies (data not shown). In contrast, ECM produced in the presence of 30 mM chlorate was devoid of mitogenic activity toward 3T3 fibroblasts (Fig. 3B). Likewise, chlorate markedly reduced (~60%) the growth promoting activity of ECM extracts toward sparsely seeded EC (data not shown). As demonstrated in Fig. 3A, EC plated on chlorate-treated ECM responded to exogenously added bFGF in a manner similar to cells plated on regular tissue culture plastic. These results indicate that sulfation is critical for the growth promoting activity of the ECM. In other experiments, ECM produced in the absence and presence of 30 mM chlorate was digested (3 h, 37°C) with 0.1 μg/ml trypsin and the amount of bFGF in the solubilized material was determined by an immunoassay (Quantikine human bFGF, R& Systems, Minneapolis, MN). The amount of bFGF in sulfate-depleted ECM was about 10-fold lower than that determined in native ECM (i.e. 11 and 121 pg of bFGF/ECM-coated 16-mm culture well, respectively). Similar results were obtained when the ECM was digested with bacterial (Flavobacterium heparinum) heparinase I (IBEX Technologies, Montreal, Canada) rather than trypsin. The heparinase-treated ECM exerted little or no mitogenic activity on vascular EC. We investigated the effect of heparin on bFGF sequestration and the growth promoting activity of ECM produced by corneal EC maintained in the absence and presence of chlorate. Heparin was included in the cell lysis solution to prevent binding of intracellular bFGF to ECM-HS when the ECM-producing cells are lysed. When 125I-bFGF was added to the lysis solution, it was found that only about 2 and 0.2% of the added bFGF was sequestered by native and sulfate-depleted ECM, respectively, during the 5-min cell lysis period (Fig. 4). Heparin (10-20 μg/ml) inhibited by about 80% the deposition of bFGF on top of ECM produced in the absence of chlorate, and there was little or no effect to heparin on the residual binding of 125I-bFGF to ECM produced in presence of chlorate (Fig. 4). We next analyzed the effect of heparin on the growth promoting activity of ECM produced in the absence and presence of chlorate. Heparin, present during the 5-min cell lysis period, had little or no effect on the growth promoting activity of native ECM toward vascular EC seeded on ECM at a low (Fig. 5) or clonal (data not shown) cell densities. Surprisingly, the mitogenic activity toward EC of ECM produced in the presence of chlorate was stimulated (1.5-4-fold, in different experiments) when heparin was included in the cell lysis solution. This stimulation was observed both when the endothelial cells were seeded directly on the ECM (Fig. 5) and when the ECM was first digested with trypsin and aliquots of the solubilized material were tested for mitogenic activity on vascular EC (data not shown). Measurements of 125I-heparin binding revealed that under the experimental conditions applied in Fig. 5, <0.5% of the heparin was bound to the ECM and there was no difference in heparin binding to ECM produced in the absence or presence of chlorate (data not shown). It has been previously demonstrated that heparin restores the ability of chlorate-treated 3T3 fibroblasts to proliferate in response to bFGF (Rapraeger et al., 1991). We investigated whether proliferation of chlorate-treated EC can be similarly restored by native ECM, in the absence and presence of added heparin. For this purpose, vascular EC were pretreated (24 h) with 30 mM chlorate and seeded in the presence of 30 mM chlorate and absence of exogenously added bFGF, on (i) tissue culture plastic, (ii) sulfate-depleted ECM, and (iii) native ECM. Heparin (1 μg/ml) was added to some of the cultures and the cells counted on day 5 after seeding. Fig. 6 demonstrates that chlorate-treated EC exhibited little or no proliferative response to he" @default.
• W2023552163 created "2016-06-24" @default.
• W2023552163 creator A5022819960 @default.
• W2023552163 creator A5024776063 @default.
• W2023552163 creator A5037824025 @default.
• W2023552163 creator A5053574488 @default.
• W2023552163 creator A5075618091 @default.
• W2023552163 date "1996-03-01" @default.
• W2023552163 modified "2023-09-28" @default.
• W2023552163 title "Sulfate Moieties in the Subendothelial Extracellular Matrix Are Involved in Basic Fibroblast Growth Factor Sequestration, Dimerization, and Stimulation of Cell Proliferation" @default.
• W2023552163 cites W1487884828 @default.
• W2023552163 cites W1494551606 @default.
• W2023552163 cites W1504197390 @default.
• W2023552163 cites W1559464451 @default.
• W2023552163 cites W1562216431 @default.
• W2023552163 cites W1578995377 @default.
• W2023552163 cites W1636802518 @default.
• W2023552163 cites W1886309537 @default.
• W2023552163 cites W1950519844 @default.
• W2023552163 cites W1974873948 @default.
• W2023552163 cites W1980982223 @default.
• W2023552163 cites W1995207257 @default.
• W2023552163 cites W1995453479 @default.
• W2023552163 cites W2006641366 @default.
• W2023552163 cites W2006868468 @default.
• W2023552163 cites W2008365877 @default.
• W2023552163 cites W2008980811 @default.
• W2023552163 cites W2011455879 @default.
• W2023552163 cites W2011610089 @default.
• W2023552163 cites W2017749217 @default.
• W2023552163 cites W2033702973 @default.
• W2023552163 cites W2034456113 @default.
• W2023552163 cites W2037170729 @default.
• W2023552163 cites W2045965305 @default.
• W2023552163 cites W2046135781 @default.
• W2023552163 cites W2050720480 @default.
• W2023552163 cites W2054703329 @default.
• W2023552163 cites W2076100222 @default.
• W2023552163 cites W2078571162 @default.
• W2023552163 cites W2086565983 @default.
• W2023552163 cites W2090341826 @default.
• W2023552163 cites W2128749862 @default.
• W2023552163 cites W2146344567 @default.
• W2023552163 cites W2156388300 @default.
• W2023552163 cites W2157756102 @default.
• W2023552163 cites W2163170931 @default.
• W2023552163 cites W2165233782 @default.
• W2023552163 cites W2225212339 @default.
• W2023552163 cites W4230053059 @default.
• W2023552163 cites W4251144546 @default.
• W2023552163 doi "https://doi.org/10.1074/jbc.271.9.4879" @default.
• W2023552163 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8617759" @default.
• W2023552163 hasPublicationYear "1996" @default.
• W2023552163 type Work @default.
• W2023552163 sameAs 2023552163 @default.
• W2023552163 citedByCount "54" @default.
• W2023552163 countsByYear W20235521632012 @default.
• W2023552163 countsByYear W20235521632013 @default.
• W2023552163 countsByYear W20235521632014 @default.
• W2023552163 countsByYear W20235521632015 @default.
• W2023552163 countsByYear W20235521632016 @default.
• W2023552163 countsByYear W20235521632017 @default.
• W2023552163 crossrefType "journal-article" @default.
• W2023552163 hasAuthorship W2023552163A5022819960 @default.
• W2023552163 hasAuthorship W2023552163A5024776063 @default.
• W2023552163 hasAuthorship W2023552163A5037824025 @default.
• W2023552163 hasAuthorship W2023552163A5053574488 @default.
• W2023552163 hasAuthorship W2023552163A5075618091 @default.
• W2023552163 hasConcept C106487976 @default.
• W2023552163 hasConcept C12554922 @default.
• W2023552163 hasConcept C134018914 @default.
• W2023552163 hasConcept C170493617 @default.
• W2023552163 hasConcept C185592680 @default.
• W2023552163 hasConcept C189165786 @default.
• W2023552163 hasConcept C202751555 @default.
• W2023552163 hasConcept C24998067 @default.
• W2023552163 hasConcept C2780381497 @default.
• W2023552163 hasConcept C28406088 @default.
• W2023552163 hasConcept C43617362 @default.
• W2023552163 hasConcept C55493867 @default.
• W2023552163 hasConcept C62112901 @default.
• W2023552163 hasConcept C74373430 @default.
• W2023552163 hasConcept C86803240 @default.
• W2023552163 hasConcept C95444343 @default.
• W2023552163 hasConceptScore W2023552163C106487976 @default.
• W2023552163 hasConceptScore W2023552163C12554922 @default.
• W2023552163 hasConceptScore W2023552163C134018914 @default.
• W2023552163 hasConceptScore W2023552163C170493617 @default.
• W2023552163 hasConceptScore W2023552163C185592680 @default.
• W2023552163 hasConceptScore W2023552163C189165786 @default.
• W2023552163 hasConceptScore W2023552163C202751555 @default.
• W2023552163 hasConceptScore W2023552163C24998067 @default.
• W2023552163 hasConceptScore W2023552163C2780381497 @default.
• W2023552163 hasConceptScore W2023552163C28406088 @default.
• W2023552163 hasConceptScore W2023552163C43617362 @default.
• W2023552163 hasConceptScore W2023552163C55493867 @default.
• W2023552163 hasConceptScore W2023552163C62112901 @default.
• W2023552163 hasConceptScore W2023552163C74373430 @default.

• next