Modulation of Vascular Human Endothelial and Rat Smooth Muscle Cell Growth by a Fucosylated Chondroitin Sulfate from Echinoderm

Summary

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC₅₀ =10 ± 5 µg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 µg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 ± 7.2%) and 27% (126.9 ± 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 µg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 ± 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 ± 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form, +98% (198.2 ± 25.%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Key words

Sulfated polysaccharides - endothelial cells - smooth muscle cells - vascular cell growth - growth factors

• References

• 1 Miao HQ, Ishai-Michaeli R, Peretz T, Vlodavsky I. Laminarin sulfate mimics the effects of heparin on smooth muscle cell proliferation and basic fibroblast growth factor-receptor binding and mitogenic activity. J Cell Physiol 1995; 164: 482-90.
• 2 Hoffman R, Paper DH, 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; 108: 3591-8.
• 3 Hoffman R, Paper DH, Donaldson J, Vogl H. Inhibition of angiogenesis and murine tumour growth by laminarin sulphate. Brit J Cancer 1996; 73: 1183-6.
• 4 Logeart D, Prigent-Richard S, Jozefonvicz J, Letourneur D. Fucans, sulfated polysaccharides extracted from brown seaweeds, inhibit vascular smooth muscle cell proliferation. I. Comparison with heparin for antiproliferative activity, binding and internalization. Eur J Cell Biol 1997; 74: 376-84.
• 5 Giraux JL, Matou S, Bros A, Tapon-Bretaudiere J, Letourneur D, Fischer AM. Modulation of human endothelial cell proliferation and migration and migration by fucoidan and heparin. Eur J Cell Biol 1998; 77: 352-9.
• 6 Santos JA, Mulloy B, Mourão PAS. Structural diversity among sulfated α-L-galactans from ascidians (Tunicates): studies on the species Ciona intestinalis and Herdmania monus. Eur J Biochem 1992; 204: 669-77.
• 7 Pavão MSG, Mourão PAS, Mulloy B, Tollefsen DM. A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on anticoagulant activity. J Biol Chem 1995; 270: 31027-36.
• 8 Nagase H, Enjyoji K, Minamiguchi K, Kitazato KT, Kitazato K, Saito H, Kato H. Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactorII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. Blood 1995; 85: 1527-34.
• 9 Alves AP, Mulloy B, Diniz J, Mourão PAS. Sulfated polysaccharides from the egg jelly are species-specific inducers of acrosomal reaction in sperm of sea urchins. J Biol Chem 1997; 272: 6965-71.
• 10 Pavão MSG, Aiello KRM, Werneck CC, Silva LCF, Valente AP, Mulloy B, Colwell NS, Tollefsen DM, Mourão PAS. Highly sulfated dermatan sulfates from ascidians: structure versus anticoagulant activity of these glycosaminoglycans. J Biol Chem 1998; 273: 27848-57.
• 11 Mourão PAS, Pereira MS, Pavão MS, Mulloy B, Tollefsen DM, Mowinckel MC, Abildgaard U. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. J Biol Chem 1996; 271: 23973-84.
• 12 Mourão PA, Guimaraes MA, Mulloy B, Thomas S, Gray E. Antithrombotic activity of a fucosylated chondroitin sulfate from echinoderm: sulfated fucose branches on the polysaccharide account for its antithrombotic action. Br J Haematol 1998; 101: 647-52.
• 13 Vieira RP, Mourão PAS. Occurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucumber. J Biol Chem 1988; 263: 18176-83.
• 14 Vieira RP, Mulloy B, Mourão PAS. Structure of a fucose-branched chondroitin sulfate from sea cucumber: evidence for the presence of 3-Osulfo-β-D-glucuronosyl residues. J Biol Chem 1991; 266: 13530-6.
• 15 Gospodarowicz D, Massoglia S, Cheng J, Fujii DK. Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cell derived from bovine adrena cortex, brain cortex and corpus luteum capillaries. J Cell Physiol 1986; 127: 121-36.
• 16 Haemmerle H, Betz E, Herr D. Human endothelial cells are stimulated and vascular smooth muscle cells are inhibited in their proliferation and migration by heparins. Vasa 1991; 20: 207-14.
• 17 Klein-Soyer C, Beretz A, Cazenave JP, Winttendorp-Rechenmann E, Vonesch JL, Rechenmann RV, Driot F, Maffrand JP. Sulfated polysaccharides modulate effects of acidic and basic fibroblast growth factors on repair of injured confluent human vascular endothelium. Arteriosclerosis 1989; 09: 147-53.
• 18 Clowes AW, Karnovsky MJ. Suppression by heparin of smooth muscle cell proliferation in the injured arteries. Nature 1997; 265: 625-36.
• 19 Guyton JR, Rosenberg RD, Clowes AW, Karnovsky MJ. Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and non anticoagulant heparin. Circ Res 1980; 46: 625-34.
• 20 Hoover RL, Rosenberg RD, Hearing W, Karnovsky MJ. Inhibition of rat arterial smooth muscle cell proliferation by heparin. II: in vitro studies. Circ Res 1980; 47: 578-83.
• 21 Reilly CF, Fritze LMS, Rosenberg RD. Heparin inhibition of smooth muscle cell proliferation. J Cell Physiol 1986; 129: 11-9.
• 22 Mourão PAS, Bastos IG. Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea. Eur J Biochem 1987; 166: 639-45.
• 23 Letourneur D, Logeart D, Avramoglou T, Jozefonvicz J. Antiproliferative capacity of synthetic dextrans on smooth muscle cell growth: The model of derivatized dextrans as heparin-like polymers. J Biomater Sci Polym 1993; 04: 431-44.
• 24 Letourneur D, Caleb BL, Castellot JJ. Heparin binding, internalization and metabolism in vascular smooth muscle cells. I: Upregulation of heparin binding correlates with antiproliferative activity. J Cell Physiol 1995; 165: 676-86.
• 25 Castellot JJ, Favreau LV, Karnovsky MJ, Rosenberg RD. Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin. A possible role of platelet endoglycosidase. J Biol Chem 1982; 257: 11256-60.
• 26 Jaffe EA, Nacheman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-6.
• 27 Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures. Anal Biochem 1986; 159: 109-13.
• 28 Giraux JL, Tapon-Bretaudiere J, Matou S, Fischer AM. Fucoidan, as heparin, induces Tissue Factor Pathway Inhibitor release from cultured human endothelial cells. Thromb Haemost 1998; 80: 692-5.
• 29 Xuereb JM, Herbert JM, Sie P, Boneu B, Constans J. Effects of heparin and related sulfated polysaccharides on tissue factor expression induced by mitogenic and non-mitogenic factors in human vascular smooth muscle cells. Thromb Haemost 1999; 81: 151-6.
• 30 Fager G, Hansson GK, Ottosson P, Dahllof B, Bondjers G. Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res 1988; 176: 319-35.
• 31 Au YP, Kenagy RD, Clowes AW. Heparin selectively inhibits the transcription of tissue-type plasminogen activator in primate arterial smooth muscle cells during mitogenesis. J Biol Chem 1992; 267: 3438-44.
• 32 Castellot JJ, Pukac LA, Caleb BL, Wright TC, Karnovsky MJ. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J Cell Biol 1989; 109: 3147-55.
• 33 Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle cells. A new model with advance differentiation. Circ Res 1999; 85: 99-107.
• 34 Rogers C, Karnovsky MJ, Edelman ER. Inhibition of experimental neointimal hyperplasia and thrombosis depends on the type of vascular injury and the site of drug administration. Circulation 1993; 88: 1215-21.
• 35 Pukac LA, Hirsch GM, Lormeau JC, Petitou M, Choay J, Karnovsky MJ. Antiproliferative effects of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo. Am J Pathol 1991; 139: 1501-9.
• 36 Rosenbaum J, Tobelem G, Molho P, Bârzu T, Caen JP. Modulation of endothelial cell growth induced by heparin. Cell Biol Int Rep 1986; 10: 437-46.
• 37 Kimura I, Nagaura T, Naitoh T, Kobayashi S, Kimura M. Heparin inhibits the progression phase of subcultured endothelial cell proliferation in rat aorta. Jpn J Pharmacol 1992; 60: 369-75.
• 38 Imamura T, Mitsui Y. Heparan sulfate and heparin as a potentior or a suppressor of growth of normal and transformed vascular endothelial cells. Exp Cell Res 1987; 172: 92-100.
• 39 Yayon A, Klagsburn M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991; 64: 841-8.
• 40 Gospodarowicz D, Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol 1986b; 128: 475-84.
• 41 Sato Y, Rifkin DB. Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 1988; 107: 1199-205.
• 42 Herbert JM, Cottineau M, Driot F, Pereillo JM, Maffrand JP. Activity of pentosan polysulphate and derivated compounds on vascular endothelial cell proliferation and migration induced by acidic and basic FGF in vitro. Biochem Pharmacol 1988; 37: 4281-8.
• 43 Hinz U, Grulich-Henn J, Preissner KT, Müller-Berghaus G. Mechanism of extrinsic pathway inhibitor stimulation by heparin in umbilical vein endothelial cells. Thromb Haemost 1991; 66: 952.
• 44 Lupu C, Kanthou C, Lupu F, Kakkar VV. Heparin induces release of intracellular tissue pathway inhibitor from human endothelial cells in culture. Thromb Haemost 1997; (suppl): 565.
• 45 Nagase H, Enjyoji K, Kamikubo Y, Kitazato KT, Kitazato K, Saito H, Kato H. Effect of Depolymerized holothurian glycosaminoglycan (DHG) on tissue factor pathway inhibitor: in vitro and in vivo studies. Thromb Haemost 1997; 78: 864-70.
• 46 Ross R. The pathogenesis of atherosclerosis - An update. N Engl J Med 1986; 314: 488-500.
• 47 Schmidt A, Yoshida K, Buddecke E. The antiproliferative activity of arterial heparan sulfate resides in domains enriched with 2-O-sulfated uronic acid residues. J Biol Chem 1992; 267: 19242-7.