- Email: [email protected]
Specificity of glycosaminoglycan suppression of endothelin-1 production by human umbilical vein endothelial cells
Life Scii
PI1 SOO24-32OS(!M)OO246-5
ELSEVIER
Vol. 65, No. 3. pp. 279-284.1999 C+Ti&tQ1999ElsaviaSCiOIlLXhC. PrintedinmSUSA. Allrigtmrem’ed 0024-32WS9/S-sm hat matter
SPECIFICITY OF GLYCOSAMINOGLYCAN SUPPRESSION OF ENDOTHFAIN-1 PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
Alice 0. Valencia, Mayia M. Mileva, Harry S. Dweck*, Louis Rosenfeld Neonatal Research Laboratory, Neonatal Division, Department of Pediatrics, New York Medical College, Westchester County Medical Center, Valhalla, NY (Received in final form March 22, 1999)
Summary
Endothelin-1 (ET-l) is the most potent vasoconstrictor peptide found in nature. Its production is stimulated by thrombin. By inhibiting thrombin we have previously shown that heparin, a highly negatively-charged glycosaminoglycan (GAG), suppresses the production of ET-l by cultured human umbilical vein endothelial cells @IUVEC). The purpose of our study is to determine the effect of other GAGS and related compounds on ET-l production. The GAGS and related compounds used in the study were: chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, fucoidin, low molecular weight dextran sulfate, high molecular weight dextran sulfate, and hyaluronan. HWEC were incubated for 48 hr with media containing these GAGS and related compounds and with media without GAG as control. ET-1 levels were measured by radioimmunoassay. GAGS and related molecules with higher sulfate content, heparin, chondroitin sulfate B, low and high molecular weight dextran sulfates significantly suppressed ET-1 production by HUVEC. Fucoidin also suppressed ET-l production despite its lower sulfate content, probably because of its structural similarity to heparin. These compounds may be useful for future in vivo studies. Key Words:endothelin-1, glyd
noglycan, human umbilical vein endothelial cells
Endothelin-1 (ET-l) is the most potent vasoconstrictor peptide currently known in nature (1). ET-l is endowed with other important biologic actions and it imparts different pathologic conditions in various tissues (2). In the cardiovascular system, ET-l has positive chronotropic and inotropic effects (3,4,5). It may be involved in hypertension (6,7), myocardial infarction (8,9,10), angina pectoris (10-13) and heart failure (14-18). There are reports that ET-l is involved in the closure of the ductus arteriosus (2,19). In the pulmonary system, ET-l possibly induces bronchoconstriction (2,20) that may contribute to the development of pulmonary hypertension and asthma (21). In the kidney, ET-l induces various glomerular, tubular and endocrinological effects (2,22). In the central nervous system, ET-1 may contribute to the vasospasm associated with subarachnoid hemorrhage (2,23). These biologic and pathologic Correspondence to: Louis Rosenfeld, Ph.D., Division of Neonatology, Department of Pediatrics, New York Medical College, Valhalla, NY 10595; Phone: (914)594-4621; Fax: (914)594-4660; E-mail: [email protected]. *Deceased
280
GlycosaminoglycanSuppressionof ET-l
Vol. 65, No. 3, 1999
effects of ET-l are mediated by at least two different receptors ETA and ETn (24) that are encoded by different genes and are characterized by different tissue distribution and pharmacological properties (25-3 1). Heparin is a heterogeneous mixture of glycosaminoglycan (GAG) chains and is widely used as an anticoagulant and antithrombotic agent (32). It is a sulfated alternating copolymer of glucosamine and iduronic or glucuronic acid residues. Heparin suppresses ET-l production in endothelial cells possibly by inhibiting a thrombin-stimulated protein kinase C-dependent signaling pathway (33) resulting in lower steady state preproendothelin mRNA levels. Other studies have shown that heparin completely blocked the release of ET-1 induced by phorbolester. Heparin also partially inhibited ET-l production stimulated by angiotensin and vasopressin (34). Our group had previously demonstrated that the suppressive effect of heparin on ET-1 production is independent of heparin molecular size, but is dependent on heparin charge and anticoagulant activity (32,35). In addition, the suppressive effect of heparin on ET-l production is time and concentration dependent up to 10 &ml (32,35) above which there is no concentration effect. Furthermore, we showed that the related GAG heparan sulfate also suppressed ET-l production to the same extent as heparin (32). The use of heparin to study ET-l and its vasopressive properties in vivo would be overshadowed by heparin’s anticoagulant and antithrombotic properties. Thus, in the present study we aimed to determine whether glycosaminoglycans (GAGS) and related compounds other than heparin can suppress the production of ET-1 in cultured human umbilical vein endothelial cells (HUVEC). Materials and Methods Human Umbilical Vein Endothelial Cells (HUVEC). Primary HLJVEC cultures were grown as previously described (32). Briefly, HUVEC were obtained by digestion of cannulated veins of umbilical cords with collagenase (Boehringer Mannheim, Indianapolis, IN). The cells were grown in complete culture medium, containing M-199 (Sigma, St. Louis, MO), 10% fetal bovine serum (Sigma), antibiotics (penicillin and streptomycin, Gibco, Grand Island, NY), Fungizone (Gibco), endothelial cell growth factor (ECGF) and heparin (Sigma, porcine, 181 units/ml). Growth was at 37 “C in a humidified atmosphere of 5% COZ. Culture medium was replaced once every 48-72 hours. Before incubation, cells were grown in heparin-free media for at least one passage. Incubations. All incubations were at 37°C in a humidified atmosphere of 5% CO2 (32). After the cells were passaged to 24-well plates in heparin-free medium, they were allowed to become confluent overnight. Control wells were given fresh media without GAG at the beginning of the incubation period. To start the incubation, the medium in each well was replaced with 1 ml of fresh culture medium containing GAG. The media were not changed for the duration of the experiment (48 h.). GAGS tested included heparin, chondroitin sulfate A (porcine rib cartilage, Sigma), chondroitin sulfate B (porcine intestine, Sigma), chondroitin sulfate C (shark cartilage, Sigma) and hyaluronan at 90 &ml each. In a separate experiment, heparin, low and high molecular weight dextran sulfates and fucoidin were incubated at 50 &ml. Previous studies with heparin showed there was no concentration dependency on this effect above 10 &ml (32). Since heparin at both concentration levels suppressed ET-l the same percentage, data from the two concentration levels were combined for analysis. For multiple 24-well plate experiments, all of the different experimental conditions were present on each plate. Each
281
Glycosaminoglycan Suppression of ET-l
Vol. 65, No. 3, 1999
experimental design was repeated at least two times. At the end of the incubation time, the medium was removed from each well, placed in tubes containing 7.5 mg potassium EDTA (Vacutainer, B&on-Dickenson, Franklin Lakes, NJ), and frozen at -70°C. Atter washing the remaining cells with Dulbecco’s phosphate-buffered saline (Gibco), NaOH solution (0.1 M) was added to each well to digest the cells for protein assay to determine cell number (32). Measurement oflmmunoreactive ET-I. Prior to assay, the thawed samples were extracted with Sep-Pak C-18 cartridges (Waters, Millipore Corporation, Milford, MA) as previously described (32). After lyophilization, the samples were reconstituted for radioimmunoassay (RIA) and aliquots were taken in duplicate for assay. Immunoreactive ET-l was measured by an RIA kit (Peninsula Laboratories, Belmont, CA) as previously described. Results were calculated as pg ET-l/100,000 cells and converted to percent of the control without GAG. Error is expressed as SEM, and significance was at a level of p < 0.05. Results Figure 1 shows relative ET-1 levels produced by I-IUVEC incubated with various GAGS. The vertical axis represents relative ET-1 concentrations, and the horizontal axis lists the GAGS that were tested. When compared to the control without GAG, represented by the solid black bar, the bars with patterns or open bars show the effects of the GAGS on relative ET-l concentrations. Heparin (I-IEP), chondroitin sulfate B (CSB), low-molecular weight and highmolecular weight dextran sulfates (LDS and I-IDS) and fucoidin (FCN) represent the GAGS which significantly suppressed ET-l production by HUVEC. CSA, CSC and HA are the GAGS that did not suppress the production ET- 1.
of
120
=
w-9
100
g
60
z
60
y
40
Iii -
20
s
T
a L
0
CONTROL (No GAG)
HEP
CSB
LDS
HDS
FCN
CSA
CSC
4
1 HA
Fig. 1 Effect of GAGS on suppression of ET-l by HUVEC. The GAGS tested are shown on the horizontal axis, and the ET-l levels (mean f SEM), relative to the control (solid bar), are shown on the vertical axis. GAGS that significantly suppress ET-l production are represented by bars with the diagonal pattern, and those that do not suppress ET-l are shown by open bars. Sulfate content for each GAG, in weight percent, is shown in parentheses above each bar (37). *p < 0.05; **p < 0.01.
282
Glycosamimglycan Suppressionof ET- 1
Vol. 65, No. 3, 1999
To ascertain whether suppression of ET-l production is related to the sulfate content of the GAGS, values for sulfate content (37) are listed in parentheses above each bar in Fig. 1. Sulfate values for dextran sulfate and fucoidin were obtained Born the manufacturer (Sigma). Heparin, chondroitin sulfate B, and dextran sulfates have high sulfate content and significantly suppressed ET-l production. Although fir&din contains a low sulfate content, it was still able to significantly suppress ET-1 production, possibly because its three-dimensional structure is similar to heparin. GAGS that do not suppress ET-1 production and have low sulfate content are CSA, CSC and HA. Discussion
Many agents have been found to stimulate and inhibit the synthesis and release of ET-l. ET-1 was initially isolated from conditioned media of cultured porcine aortic endothelial cells. It consists of 21 amino acid residues and contains two intramolecular disulfide bonds (1). There are three isotypes of endothelin ET-I, ET-2 and ET-3 made from different gene products (38). ET-1 is the only endothelin produced by endothelial cells (2,24). It is also produced in vascular smooth muscle cells (2). In humans, the ET-l gene is located on chromosome 6 (1). This gene produces the precursor prepro-ET-l (203 amino acids) which is processed to a 38 amino acid propeptide called big ET-l. Big ET-l is the precursor for ET-l. The mechanism of the production and release of ET-l from endothelial cells remains unclear. It is generally believed that the synthesis and secretion is regulated at the step of the synthesis of the prepro-ET-l by transcription of the ET-l gene. Vasoactive factors which stimulate the synthesis and secretion of ET-l include thrombin, tumor necrosis factor-g, interleukin-1 and -6, ionomycin, angiotensin II, arginine, vasopressin, calcium ionophores, insulin, oxidized low-density lipoproteins, high-density lipoproteins, hypoxia, &hernia and shear stress (1,24,39-42). The production and release of ET-l is inhibited by atrial natriuretic peptides, endothelium-derived relaxing factor, nitroglycerin, prostaglandin Ez, prostaglandin 1~ and heparin (34,39,43-46). Among these inhibitory agents, heparin’s suppressive effect on the production of ET-l has been widely studied (32,34,35,46). No previous studies have investigated the effect of other GAGS in the production of ET- 1. Glycosaminoglycans are a family of structurally distinct polyanionic, complex carbohydrates that include heparin, heparan sulfate, chondroitin sulfate A chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, and hyaluronic acid (47). The polysaccharide chains are composed almost entirely of repeating disaccharide units consisting of an amino sugar (either D-glucosamine or galactosamine) and uranic acid. In addition, related compounds include the dextran sulfates (a polymer of sulfated glucose units) (47), and fircoidin (a polymer containing sulfated ficose units) (48). In conclusion, our study demonstrates that, in addition to heparin, chondroitin sulfate B, fiicoidin, and low and high molecular weight dextran sulfates also suppress the production of ET-l. Suppression of ET- 1 by glycosaminoglycans is dependent on their high sulfate content or their structural similarity to heparin. These compounds have lower anticoagulant and antithrombotic properties than heparin and may be better suited for in vivo studies on pulmonary or systemic hypertension. Acknowledgements
We wish to thank the nursing staffs at Westchester Medical Center and Phelps Memorial Hospital for supplying fresh umbilical cords for this study.
Vol. 65, No. 3, 1999
Glycosaminoglycan
Suppnssion
of ET-l
283
References S. KIMURA, Y. TOMOBE, M. KOBAYASHI, Y. 1. M. YANAGISAWA, H. KuRMARq MITSUI, Y. YAZAKI, K. GOTO, and T. MASAKI, Nature 332 411-415 (1988). 2. E.R. LEVIN, N. Engl. J. Med. 333 356-363 (1995). 3. C.S. MORAVEC, E.E. REYNOLDS, R.W. STEWART, and M. BOND, Biochem. Biophys. Res. Commun. 159 14-28 (1989). 4. T. ISHIKAWA, M. YANAGISAWA, S. KIMURA, K. GOTO, and T. MASAKI, Am. 1. Physiol. 255 H970-973 (1988). 5. J.R. HU, R. VON HARSDORF, and R.E. LANG, Eur. J. Pharmacol. 158 275-278 (1988). 6. K.L. GOETZ, B.C. WANG, J.B. MADWED, J.L. ZHU, and R.J. LEADLEY, Am. 1. Physiol. 16 235-238 (1989). 7. M. CLOZEL, V. BREU, L. BURRI, J.M. CASSAL, W. FISCHLI, G.A. GRAW, G. HIRTH, and B.M. LOFFLER, Nature 365 759-761 (1993). 8. K. SALMIEN, I. TIKKANEN, 0. SAIJONMAA, M. NIEMINEN, F. FYHRQUIST and M. FRICK, Lancet (letter) 2 747 (1989). 9. T. MIYAUCHI, M. YANAGISAWA, T. TOMlZAWA, Y. SUGISHITA, N. SUZUKI, M. FUJINO, R. AJISAKA, K. GOTO, and T. MASAKI, Lancet (letter) 2 53-54 (1989). 10. I. WIECZOREK, W.G. HAYNES, D.J. WEBB, CA. LUDLAM, and K.A.A. FOX, Br. Heart J. 72 436-441 (1994). 11. A.M. ZEIHER H. GOEBEL, V. SCHACI-IINGER, and C. RILING, Circulation 91 941947 (1995). 12. J. PERNOW, and Q. WANG, Cardiovasc. Res. 33 5 15-526 (1997). 13. T. TOYOOKA, T. AIZAWA, N. SUZUKI, Y. HIRATA, T. MIYAUCHI, W.S. SHIN, M. YANAGISAWA, T. MASAKI and T. SUGIMOTO, Circulation 83 476-483 (1991). 14. T. OMLAND, R.T. LIE, A. AAKVAAG, T. AARSLAND, and K. DICKSTEIN, Circulation 89 1573-1579 (1994). 15. J.R. TEERLINK, BM. LOFFLER, P. HESS, J.-P. MAIRE, M. CLOZEL, and J.-P. CLOZEL, Circulation 90 25 10-25 18 (1994). 16. R.J. RODEHEFFER A. LERMAN, D. M. HEUBLEIN, and J.C. BURNETT, JR., Mayo Clinic Proceedings 67 719-724 (1992). 17. CM. WEI, A. LERMAN, R.J. RODEHEFFER, C.G.A. MCGREGOR R.R. BRANDT, S. WRIGHT, D.M. HEUBLEIN, P.C. KAO, W.D. EDWARDS, and J.C. BURNETT, JR., Circulation 89 1580-l 586 (1994). 18. R. PACHER, J. BERGLER-KLEIN, S. GLOBITS, H. TEUFELSBAUER, M. SCHULLER, A. KRAUTER, E. ORGIS, S. RODLER, M. WUTTE, and E. HARTTER, Am. J. Cardiol. 71 1293-1299 (1993). 19. F. COCEANI, L. KELSEY, and E. SEIDLITZ, Can. J. Physiol. Pharmacol. 70 1061-1064 (1992). 20. G.M. RUBANYI, Pharmacol. Rev. 46 345-415 (1994). 21. D. W. P. HAY, P. J. HENRY, and R. G. GOLDIE, Am. J. Respir. Crit. Care Med. 154 1594-1597 (1996). 22. V. KON, T. YOSHIOKA, A. FOGG, and I. ICHIKAWA, J. Clin. Invest. 83 1762-1767 (1989). 23. R.N. WILLETTE, H. ZHANG, M.P. MITCHELL, C.F. SAUERMELCH, E.H. OHLSTEIN, and A.C. SULPIZIO, Stroke 25 2450-2456 (1994). 24. H. KANAIDE, Gen. Pharmac. 27 559- 63 (1996). 25. Y. OGAWA, K. NAKAO, H. ARAI, 0. NAKAGAWA, K. HOSODA S. SUGA, S. NAKANISHI, and H. IMUR4, Biochem. Biophys. Res. Cummun. 178 248-255 (1991). 26. H.Y. LIN, E.H. KAJI, G.K. WINKEL, H.E. IVES, and H.F. LODISH, Proc. Natl. Acad. Sci. U.S.A. 88 3185-3189.
284
Glycosaminoglycan Suppression of ET-I
Vol. 65, No. 3, 1999
27. H. ARAI, S. HORI, I. ARAMORI, H. OHKUBO, and S. NAKANISHI, Nature 348 730735 (1990). 28. T. SAKURAI, M. YANAGISAWA, and T. MASAKI, Trends Pharmacol. Sci. 13 103-108 (1993). 29. T. SAKURAI, M. YANAGISAWA, Y. TAKUWA, H. MIYAZAKI, S. KIMURA, K. GOTO, and T. MASAKI, Nature 348 732-735 (1990). 30. T. MASAKI, Pharmacol. Rev. 46 137-142 (1994). 31. M. CESARI, E. PAVAN, A. SACCHETTO, and G.P. ROSSI, Am. Heart J. 132 12361243 (1996). 32. S. REANTRAGOON, L. ARRIGO, M. ABOU SEOUD, H. DWECK, and L. ROSENFELD, Arch. Biochem. Biophys. 314 315-322 (1994). 33. J.W. ASSENDER, E. IRENIUS, and B.B. FREDHOLM, Acta. Physiol. Stand. 157 451460 (1996). 34. T. IMAI, Y. HIRATA, T. EMORI, and F. MARUMO, Hypertension 21353-358 (1993). 35. S. REANTRAGOON, L. M. ARRIGO, H. S. DWECK, and L. ROSENFELD, Arch. Biochem. Biophys. 327 234-238 (1996). 36. K. YOKOKAWA, M. KOHNO, H. TAHARA, A. MANDAL, K. YASUNARI, K. MURAKAWA, T. HORIO, M. IKEDA, M. MINAMI and T. TAKEDA, J. Cardiovasc. Pharmacol. 22 (Suppl. 8) S46-S48 (1993). 37. M. 0. LONGAS, and K. 0. BREITWEISER, Anal. Biochem. 192 193-196 (1991). 38. A. INOUE, M. YANAGISAWA, S. KIMURA, Y. KASUYA, T. MIYAUCHI, K. GOTO, and T. MASAKI, Proc. Natl. Acad. Sci. U.S.A. 86 2834-2867 (1989). 39. C. BOULANGER, and T.F. LUSCHER, J. Clin. Invest. 85 587-590 (1990). 40. T. MASAKI, M. YANAGISAWA, K. GOTO, and S. KIMURA, J. Hypertension 8 (Suppl. 7) S107-S112 (1990). 41. R.F. O’BRIEN, R.J. ROBBINS, and I.F. MCMURTRY, J. Cell Physiol. 132 263-270 (1987). T. SUGIYAMA, F. TAKAKU, M. YANGISAWA, T. 42. M. YOSHIZUMI, H. KuRIHARq MASAKI, and Y. YAZAKI, Biochem. Biophys. Res. Comm. 161 859-864 (1989). T. HORIO, and T. 43. M. KOHNO, K. YASUNARI, K. YOKOKAWA, K. MURAKAWA, TAKEDA, J. Clin. Invest. 87 1999-2004 (1991). 44. R.M. HU, E.R. LEVIN, A. PEDRAM, and H.J.L. FRANK, J. Biol. Chem. 267 1738417389 (1992). 45. B.A. PRINS, RM. HU, B. NAZARIO, A. PEDRAM, H.J.L. FRANK, M.A. WEBER, and E.R. LEVIN, J. Biol. Chem. 269 11938-l 1944 (1994). 46. K. YOKOKAWA, A. MANDAL, M. KOHNO, T. HORIO, K. MURAKAWA, K. YASUNARI, and T. TAKEDA, Am. J. Physiol. 263 R1035-R1041 (1992). 47. J. GALLAGHER, Biochem. J. 236 3 13-325 (1986). 48. C.G. GLABE, T. YEDNOCK, and S.D. ROSEN, J. Cell Sci. 6 475-490 (1983).
PI1 SOO24-32OS(!M)OO246-5
ELSEVIER
Vol. 65, No. 3. pp. 279-284.1999 C+Ti&tQ1999ElsaviaSCiOIlLXhC. PrintedinmSUSA. Allrigtmrem’ed 0024-32WS9/S-sm hat matter
SPECIFICITY OF GLYCOSAMINOGLYCAN SUPPRESSION OF ENDOTHFAIN-1 PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
Alice 0. Valencia, Mayia M. Mileva, Harry S. Dweck*, Louis Rosenfeld Neonatal Research Laboratory, Neonatal Division, Department of Pediatrics, New York Medical College, Westchester County Medical Center, Valhalla, NY (Received in final form March 22, 1999)
Summary
Endothelin-1 (ET-l) is the most potent vasoconstrictor peptide found in nature. Its production is stimulated by thrombin. By inhibiting thrombin we have previously shown that heparin, a highly negatively-charged glycosaminoglycan (GAG), suppresses the production of ET-l by cultured human umbilical vein endothelial cells @IUVEC). The purpose of our study is to determine the effect of other GAGS and related compounds on ET-l production. The GAGS and related compounds used in the study were: chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, fucoidin, low molecular weight dextran sulfate, high molecular weight dextran sulfate, and hyaluronan. HWEC were incubated for 48 hr with media containing these GAGS and related compounds and with media without GAG as control. ET-1 levels were measured by radioimmunoassay. GAGS and related molecules with higher sulfate content, heparin, chondroitin sulfate B, low and high molecular weight dextran sulfates significantly suppressed ET-1 production by HUVEC. Fucoidin also suppressed ET-l production despite its lower sulfate content, probably because of its structural similarity to heparin. These compounds may be useful for future in vivo studies. Key Words:endothelin-1, glyd
noglycan, human umbilical vein endothelial cells
Endothelin-1 (ET-l) is the most potent vasoconstrictor peptide currently known in nature (1). ET-l is endowed with other important biologic actions and it imparts different pathologic conditions in various tissues (2). In the cardiovascular system, ET-l has positive chronotropic and inotropic effects (3,4,5). It may be involved in hypertension (6,7), myocardial infarction (8,9,10), angina pectoris (10-13) and heart failure (14-18). There are reports that ET-l is involved in the closure of the ductus arteriosus (2,19). In the pulmonary system, ET-l possibly induces bronchoconstriction (2,20) that may contribute to the development of pulmonary hypertension and asthma (21). In the kidney, ET-l induces various glomerular, tubular and endocrinological effects (2,22). In the central nervous system, ET-1 may contribute to the vasospasm associated with subarachnoid hemorrhage (2,23). These biologic and pathologic Correspondence to: Louis Rosenfeld, Ph.D., Division of Neonatology, Department of Pediatrics, New York Medical College, Valhalla, NY 10595; Phone: (914)594-4621; Fax: (914)594-4660; E-mail: [email protected]. *Deceased
280
GlycosaminoglycanSuppressionof ET-l
Vol. 65, No. 3, 1999
effects of ET-l are mediated by at least two different receptors ETA and ETn (24) that are encoded by different genes and are characterized by different tissue distribution and pharmacological properties (25-3 1). Heparin is a heterogeneous mixture of glycosaminoglycan (GAG) chains and is widely used as an anticoagulant and antithrombotic agent (32). It is a sulfated alternating copolymer of glucosamine and iduronic or glucuronic acid residues. Heparin suppresses ET-l production in endothelial cells possibly by inhibiting a thrombin-stimulated protein kinase C-dependent signaling pathway (33) resulting in lower steady state preproendothelin mRNA levels. Other studies have shown that heparin completely blocked the release of ET-1 induced by phorbolester. Heparin also partially inhibited ET-l production stimulated by angiotensin and vasopressin (34). Our group had previously demonstrated that the suppressive effect of heparin on ET-1 production is independent of heparin molecular size, but is dependent on heparin charge and anticoagulant activity (32,35). In addition, the suppressive effect of heparin on ET-l production is time and concentration dependent up to 10 &ml (32,35) above which there is no concentration effect. Furthermore, we showed that the related GAG heparan sulfate also suppressed ET-l production to the same extent as heparin (32). The use of heparin to study ET-l and its vasopressive properties in vivo would be overshadowed by heparin’s anticoagulant and antithrombotic properties. Thus, in the present study we aimed to determine whether glycosaminoglycans (GAGS) and related compounds other than heparin can suppress the production of ET-1 in cultured human umbilical vein endothelial cells (HUVEC). Materials and Methods Human Umbilical Vein Endothelial Cells (HUVEC). Primary HLJVEC cultures were grown as previously described (32). Briefly, HUVEC were obtained by digestion of cannulated veins of umbilical cords with collagenase (Boehringer Mannheim, Indianapolis, IN). The cells were grown in complete culture medium, containing M-199 (Sigma, St. Louis, MO), 10% fetal bovine serum (Sigma), antibiotics (penicillin and streptomycin, Gibco, Grand Island, NY), Fungizone (Gibco), endothelial cell growth factor (ECGF) and heparin (Sigma, porcine, 181 units/ml). Growth was at 37 “C in a humidified atmosphere of 5% COZ. Culture medium was replaced once every 48-72 hours. Before incubation, cells were grown in heparin-free media for at least one passage. Incubations. All incubations were at 37°C in a humidified atmosphere of 5% CO2 (32). After the cells were passaged to 24-well plates in heparin-free medium, they were allowed to become confluent overnight. Control wells were given fresh media without GAG at the beginning of the incubation period. To start the incubation, the medium in each well was replaced with 1 ml of fresh culture medium containing GAG. The media were not changed for the duration of the experiment (48 h.). GAGS tested included heparin, chondroitin sulfate A (porcine rib cartilage, Sigma), chondroitin sulfate B (porcine intestine, Sigma), chondroitin sulfate C (shark cartilage, Sigma) and hyaluronan at 90 &ml each. In a separate experiment, heparin, low and high molecular weight dextran sulfates and fucoidin were incubated at 50 &ml. Previous studies with heparin showed there was no concentration dependency on this effect above 10 &ml (32). Since heparin at both concentration levels suppressed ET-l the same percentage, data from the two concentration levels were combined for analysis. For multiple 24-well plate experiments, all of the different experimental conditions were present on each plate. Each
281
Glycosaminoglycan Suppression of ET-l
Vol. 65, No. 3, 1999
experimental design was repeated at least two times. At the end of the incubation time, the medium was removed from each well, placed in tubes containing 7.5 mg potassium EDTA (Vacutainer, B&on-Dickenson, Franklin Lakes, NJ), and frozen at -70°C. Atter washing the remaining cells with Dulbecco’s phosphate-buffered saline (Gibco), NaOH solution (0.1 M) was added to each well to digest the cells for protein assay to determine cell number (32). Measurement oflmmunoreactive ET-I. Prior to assay, the thawed samples were extracted with Sep-Pak C-18 cartridges (Waters, Millipore Corporation, Milford, MA) as previously described (32). After lyophilization, the samples were reconstituted for radioimmunoassay (RIA) and aliquots were taken in duplicate for assay. Immunoreactive ET-l was measured by an RIA kit (Peninsula Laboratories, Belmont, CA) as previously described. Results were calculated as pg ET-l/100,000 cells and converted to percent of the control without GAG. Error is expressed as SEM, and significance was at a level of p < 0.05. Results Figure 1 shows relative ET-1 levels produced by I-IUVEC incubated with various GAGS. The vertical axis represents relative ET-1 concentrations, and the horizontal axis lists the GAGS that were tested. When compared to the control without GAG, represented by the solid black bar, the bars with patterns or open bars show the effects of the GAGS on relative ET-l concentrations. Heparin (I-IEP), chondroitin sulfate B (CSB), low-molecular weight and highmolecular weight dextran sulfates (LDS and I-IDS) and fucoidin (FCN) represent the GAGS which significantly suppressed ET-l production by HUVEC. CSA, CSC and HA are the GAGS that did not suppress the production ET- 1.
of
120
=
w-9
100
g
60
z
60
y
40
Iii -
20
s
T
a L
0
CONTROL (No GAG)
HEP
CSB
LDS
HDS
FCN
CSA
CSC
4
1 HA
Fig. 1 Effect of GAGS on suppression of ET-l by HUVEC. The GAGS tested are shown on the horizontal axis, and the ET-l levels (mean f SEM), relative to the control (solid bar), are shown on the vertical axis. GAGS that significantly suppress ET-l production are represented by bars with the diagonal pattern, and those that do not suppress ET-l are shown by open bars. Sulfate content for each GAG, in weight percent, is shown in parentheses above each bar (37). *p < 0.05; **p < 0.01.
282
Glycosamimglycan Suppressionof ET- 1
Vol. 65, No. 3, 1999
To ascertain whether suppression of ET-l production is related to the sulfate content of the GAGS, values for sulfate content (37) are listed in parentheses above each bar in Fig. 1. Sulfate values for dextran sulfate and fucoidin were obtained Born the manufacturer (Sigma). Heparin, chondroitin sulfate B, and dextran sulfates have high sulfate content and significantly suppressed ET-l production. Although fir&din contains a low sulfate content, it was still able to significantly suppress ET-1 production, possibly because its three-dimensional structure is similar to heparin. GAGS that do not suppress ET-1 production and have low sulfate content are CSA, CSC and HA. Discussion
Many agents have been found to stimulate and inhibit the synthesis and release of ET-l. ET-1 was initially isolated from conditioned media of cultured porcine aortic endothelial cells. It consists of 21 amino acid residues and contains two intramolecular disulfide bonds (1). There are three isotypes of endothelin ET-I, ET-2 and ET-3 made from different gene products (38). ET-1 is the only endothelin produced by endothelial cells (2,24). It is also produced in vascular smooth muscle cells (2). In humans, the ET-l gene is located on chromosome 6 (1). This gene produces the precursor prepro-ET-l (203 amino acids) which is processed to a 38 amino acid propeptide called big ET-l. Big ET-l is the precursor for ET-l. The mechanism of the production and release of ET-l from endothelial cells remains unclear. It is generally believed that the synthesis and secretion is regulated at the step of the synthesis of the prepro-ET-l by transcription of the ET-l gene. Vasoactive factors which stimulate the synthesis and secretion of ET-l include thrombin, tumor necrosis factor-g, interleukin-1 and -6, ionomycin, angiotensin II, arginine, vasopressin, calcium ionophores, insulin, oxidized low-density lipoproteins, high-density lipoproteins, hypoxia, &hernia and shear stress (1,24,39-42). The production and release of ET-l is inhibited by atrial natriuretic peptides, endothelium-derived relaxing factor, nitroglycerin, prostaglandin Ez, prostaglandin 1~ and heparin (34,39,43-46). Among these inhibitory agents, heparin’s suppressive effect on the production of ET-l has been widely studied (32,34,35,46). No previous studies have investigated the effect of other GAGS in the production of ET- 1. Glycosaminoglycans are a family of structurally distinct polyanionic, complex carbohydrates that include heparin, heparan sulfate, chondroitin sulfate A chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, and hyaluronic acid (47). The polysaccharide chains are composed almost entirely of repeating disaccharide units consisting of an amino sugar (either D-glucosamine or galactosamine) and uranic acid. In addition, related compounds include the dextran sulfates (a polymer of sulfated glucose units) (47), and fircoidin (a polymer containing sulfated ficose units) (48). In conclusion, our study demonstrates that, in addition to heparin, chondroitin sulfate B, fiicoidin, and low and high molecular weight dextran sulfates also suppress the production of ET-l. Suppression of ET- 1 by glycosaminoglycans is dependent on their high sulfate content or their structural similarity to heparin. These compounds have lower anticoagulant and antithrombotic properties than heparin and may be better suited for in vivo studies on pulmonary or systemic hypertension. Acknowledgements
We wish to thank the nursing staffs at Westchester Medical Center and Phelps Memorial Hospital for supplying fresh umbilical cords for this study.
Vol. 65, No. 3, 1999
Glycosaminoglycan
Suppnssion
of ET-l
283
References S. KIMURA, Y. TOMOBE, M. KOBAYASHI, Y. 1. M. YANAGISAWA, H. KuRMARq MITSUI, Y. YAZAKI, K. GOTO, and T. MASAKI, Nature 332 411-415 (1988). 2. E.R. LEVIN, N. Engl. J. Med. 333 356-363 (1995). 3. C.S. MORAVEC, E.E. REYNOLDS, R.W. STEWART, and M. BOND, Biochem. Biophys. Res. Commun. 159 14-28 (1989). 4. T. ISHIKAWA, M. YANAGISAWA, S. KIMURA, K. GOTO, and T. MASAKI, Am. 1. Physiol. 255 H970-973 (1988). 5. J.R. HU, R. VON HARSDORF, and R.E. LANG, Eur. J. Pharmacol. 158 275-278 (1988). 6. K.L. GOETZ, B.C. WANG, J.B. MADWED, J.L. ZHU, and R.J. LEADLEY, Am. 1. Physiol. 16 235-238 (1989). 7. M. CLOZEL, V. BREU, L. BURRI, J.M. CASSAL, W. FISCHLI, G.A. GRAW, G. HIRTH, and B.M. LOFFLER, Nature 365 759-761 (1993). 8. K. SALMIEN, I. TIKKANEN, 0. SAIJONMAA, M. NIEMINEN, F. FYHRQUIST and M. FRICK, Lancet (letter) 2 747 (1989). 9. T. MIYAUCHI, M. YANAGISAWA, T. TOMlZAWA, Y. SUGISHITA, N. SUZUKI, M. FUJINO, R. AJISAKA, K. GOTO, and T. MASAKI, Lancet (letter) 2 53-54 (1989). 10. I. WIECZOREK, W.G. HAYNES, D.J. WEBB, CA. LUDLAM, and K.A.A. FOX, Br. Heart J. 72 436-441 (1994). 11. A.M. ZEIHER H. GOEBEL, V. SCHACI-IINGER, and C. RILING, Circulation 91 941947 (1995). 12. J. PERNOW, and Q. WANG, Cardiovasc. Res. 33 5 15-526 (1997). 13. T. TOYOOKA, T. AIZAWA, N. SUZUKI, Y. HIRATA, T. MIYAUCHI, W.S. SHIN, M. YANAGISAWA, T. MASAKI and T. SUGIMOTO, Circulation 83 476-483 (1991). 14. T. OMLAND, R.T. LIE, A. AAKVAAG, T. AARSLAND, and K. DICKSTEIN, Circulation 89 1573-1579 (1994). 15. J.R. TEERLINK, BM. LOFFLER, P. HESS, J.-P. MAIRE, M. CLOZEL, and J.-P. CLOZEL, Circulation 90 25 10-25 18 (1994). 16. R.J. RODEHEFFER A. LERMAN, D. M. HEUBLEIN, and J.C. BURNETT, JR., Mayo Clinic Proceedings 67 719-724 (1992). 17. CM. WEI, A. LERMAN, R.J. RODEHEFFER, C.G.A. MCGREGOR R.R. BRANDT, S. WRIGHT, D.M. HEUBLEIN, P.C. KAO, W.D. EDWARDS, and J.C. BURNETT, JR., Circulation 89 1580-l 586 (1994). 18. R. PACHER, J. BERGLER-KLEIN, S. GLOBITS, H. TEUFELSBAUER, M. SCHULLER, A. KRAUTER, E. ORGIS, S. RODLER, M. WUTTE, and E. HARTTER, Am. J. Cardiol. 71 1293-1299 (1993). 19. F. COCEANI, L. KELSEY, and E. SEIDLITZ, Can. J. Physiol. Pharmacol. 70 1061-1064 (1992). 20. G.M. RUBANYI, Pharmacol. Rev. 46 345-415 (1994). 21. D. W. P. HAY, P. J. HENRY, and R. G. GOLDIE, Am. J. Respir. Crit. Care Med. 154 1594-1597 (1996). 22. V. KON, T. YOSHIOKA, A. FOGG, and I. ICHIKAWA, J. Clin. Invest. 83 1762-1767 (1989). 23. R.N. WILLETTE, H. ZHANG, M.P. MITCHELL, C.F. SAUERMELCH, E.H. OHLSTEIN, and A.C. SULPIZIO, Stroke 25 2450-2456 (1994). 24. H. KANAIDE, Gen. Pharmac. 27 559- 63 (1996). 25. Y. OGAWA, K. NAKAO, H. ARAI, 0. NAKAGAWA, K. HOSODA S. SUGA, S. NAKANISHI, and H. IMUR4, Biochem. Biophys. Res. Cummun. 178 248-255 (1991). 26. H.Y. LIN, E.H. KAJI, G.K. WINKEL, H.E. IVES, and H.F. LODISH, Proc. Natl. Acad. Sci. U.S.A. 88 3185-3189.
284
Glycosaminoglycan Suppression of ET-I
Vol. 65, No. 3, 1999
27. H. ARAI, S. HORI, I. ARAMORI, H. OHKUBO, and S. NAKANISHI, Nature 348 730735 (1990). 28. T. SAKURAI, M. YANAGISAWA, and T. MASAKI, Trends Pharmacol. Sci. 13 103-108 (1993). 29. T. SAKURAI, M. YANAGISAWA, Y. TAKUWA, H. MIYAZAKI, S. KIMURA, K. GOTO, and T. MASAKI, Nature 348 732-735 (1990). 30. T. MASAKI, Pharmacol. Rev. 46 137-142 (1994). 31. M. CESARI, E. PAVAN, A. SACCHETTO, and G.P. ROSSI, Am. Heart J. 132 12361243 (1996). 32. S. REANTRAGOON, L. ARRIGO, M. ABOU SEOUD, H. DWECK, and L. ROSENFELD, Arch. Biochem. Biophys. 314 315-322 (1994). 33. J.W. ASSENDER, E. IRENIUS, and B.B. FREDHOLM, Acta. Physiol. Stand. 157 451460 (1996). 34. T. IMAI, Y. HIRATA, T. EMORI, and F. MARUMO, Hypertension 21353-358 (1993). 35. S. REANTRAGOON, L. M. ARRIGO, H. S. DWECK, and L. ROSENFELD, Arch. Biochem. Biophys. 327 234-238 (1996). 36. K. YOKOKAWA, M. KOHNO, H. TAHARA, A. MANDAL, K. YASUNARI, K. MURAKAWA, T. HORIO, M. IKEDA, M. MINAMI and T. TAKEDA, J. Cardiovasc. Pharmacol. 22 (Suppl. 8) S46-S48 (1993). 37. M. 0. LONGAS, and K. 0. BREITWEISER, Anal. Biochem. 192 193-196 (1991). 38. A. INOUE, M. YANAGISAWA, S. KIMURA, Y. KASUYA, T. MIYAUCHI, K. GOTO, and T. MASAKI, Proc. Natl. Acad. Sci. U.S.A. 86 2834-2867 (1989). 39. C. BOULANGER, and T.F. LUSCHER, J. Clin. Invest. 85 587-590 (1990). 40. T. MASAKI, M. YANAGISAWA, K. GOTO, and S. KIMURA, J. Hypertension 8 (Suppl. 7) S107-S112 (1990). 41. R.F. O’BRIEN, R.J. ROBBINS, and I.F. MCMURTRY, J. Cell Physiol. 132 263-270 (1987). T. SUGIYAMA, F. TAKAKU, M. YANGISAWA, T. 42. M. YOSHIZUMI, H. KuRIHARq MASAKI, and Y. YAZAKI, Biochem. Biophys. Res. Comm. 161 859-864 (1989). T. HORIO, and T. 43. M. KOHNO, K. YASUNARI, K. YOKOKAWA, K. MURAKAWA, TAKEDA, J. Clin. Invest. 87 1999-2004 (1991). 44. R.M. HU, E.R. LEVIN, A. PEDRAM, and H.J.L. FRANK, J. Biol. Chem. 267 1738417389 (1992). 45. B.A. PRINS, RM. HU, B. NAZARIO, A. PEDRAM, H.J.L. FRANK, M.A. WEBER, and E.R. LEVIN, J. Biol. Chem. 269 11938-l 1944 (1994). 46. K. YOKOKAWA, A. MANDAL, M. KOHNO, T. HORIO, K. MURAKAWA, K. YASUNARI, and T. TAKEDA, Am. J. Physiol. 263 R1035-R1041 (1992). 47. J. GALLAGHER, Biochem. J. 236 3 13-325 (1986). 48. C.G. GLABE, T. YEDNOCK, and S.D. ROSEN, J. Cell Sci. 6 475-490 (1983).