Archives of Biochemistry and Biophysics Vol. 370, No. 2, October 15, pp. 176 182, 1999 Article ID abbi.1999.

1395, available online at http://www.idealibrary.com on

Inhibition of Hyaluronidase by Fully O-Sulfonated Glycosaminoglycans

Toshihiko Toida, 1 Yoshiaki Ogita, Atsushi Suzuki, Hidenao Toyoda, and Toshio Imanari
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba, Japan 2638522

Received March 1, 1999, and in revised form July 15, 1999

We report a new ow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using uorometric detection with the uorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were conrmed by 1H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC 50 values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects. 1999 Academic Press Key Words: hyaluronidase; ow injection assay; urinary hyaluronidase; fully O-sulfonated glycosaminoglycans; inhibitory effects.

Hyaluronidases (EC 3.2.1.35), which are known as one of enzymes involved in inammatory reactions, usually exists in an inactive form that is activated by metal ions and other agents (1). These enzymes are a family of -1-4-endoglucosaminidases that degrade hyaluronan (HA) 2 to a lesser extent, other glycosamiTo whom correspondence should be addressed. Fax: 81-43-2903021. E-mail: toida@p.chiba-u.ac.jp. 2 Abbreviations used: HAase, hyaluronidase; GAG(s), glycosaminoglycan(s); GlcAp, D-glucuronic acid; GlcNpAc, D-galactosamine; Ac, 176
1

noglycans (2, 3). These enzymes have been relatively neglected in comparison to other glycosidases, probably due to the lack of simple, sensitive assays that measure degradation of their polymeric substrate (4). HAases and their substrate are becoming increasingly prominent in many elds of biochemistry (5, 6). A key role for this enzyme has been recognized in a number of basic biological processes, such as embryogenesis (7, 8), carcinogenesis (9, 10), wound healing (11), angiogenesis (12), and inammation (13, 14). Clinically, aberrations of HA metabolism are associated with processes such as adult respiratory distress syndrome (15) and organ transplant edema and rejection (16, 17) and as a marker for cancer remission and relapse (18). An inherited disorder involving serum HAase deciency has been described recently (19). The HAase assay described here might be useful as a routine clinical laboratory method, as a screening method for antitumor medicines, or in the investigation of new HAase inhibitors. In addition, it has been reported that anti-allergic compounds such as tranilast (20), traxonox (1), sodium cromoglycate (21), and baicalein phosphate (1) have anti-hyaluronidase activity. Until recently, the most commonly used HAase assays were based on the measurement of the generation of new reducing N-acetylglucosamido groups (22), or a decrease in viscosity (23) or turbidity (24). Such assays are often either insensitive or lack specicity. More recently, a new generation of microtiter-based assays has been developed (25). Although these microtiterbased assays do not require special reagents, the assay procedure is complicated and requires skillful use to obtain accurate data. We have found that 2-cyanoacet-

acetyl; PAGE, polyacrylamide gel electrophoresis; GPC, gel permeation chromatography; FIA, ow injection analysis; HA, hyaluronen; TRU, turbidity reducing unit; PTFE, polytetrauoroethylene; AGA, 7-amino-1,3-naphthalenedisulfonic acid.
0003-9861/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Schematic ow diagram of the FIA system.

amide is a highly sensitive uorogenic reagent for the determination of mono-, oligo-, and polysaccharides in HPLC when coupled with postcolumn detection (26). In addition, the uorometric response of each saccharide in the detection system is dependent on the number of the reducing end groups that are present (27). These ndings suggest the application of this detection system for assay of enzymatic activity. In this paper, we have established a new ow injection analysis (FIA) method for determining HAase activity and have applied the method for the analysis of human urinary hyaluronidase activity and to measure HAase inhibitory activity of chemicallyO-sulfonated glycosaminoglycans.
MATERIALS AND METHODS
Chemicals and reagents. HA prepared from Streptococcus zooepidemicus containing HA synthetase was purchased from Kibun Chemipha (Tokyo, Japan). Chondroitin sulfate from bovine tracheal cartilage was generously donated by Shin Nippon Yakugyo Co. (Tokyo, Japan). Unsaturated disaccharides [2-acetamido-2-deoxy-3-O-( -Dgluco-4-enepyranosyluronic acid)-D-glucose ( Di-HA), 2-acetamido-2deoxy-3-O-( -D-gluco-4-enepyranosyluronic acid)-D-galactose ( Di-0S), 2-acetamido-2-deoxy-3-O-( -D-gluco-4-enepyranosyluronic acid)-6-O-sulfoD-galactose ( Di-6S), 2-acetamido-2-deoxy-3-O-( -D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose ( Di-4S), 2-acetamido-2-deoxy-3O-(2-O-sulfo- -D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose ( Di-S B)], bovine testicular HAase (Sigma type I-S), chondroitin sulfate lyase ABC (Chase ABC, EC 4.2.2.4), and ACII arthro (Chase ACII, EC 4.2.2.5) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). All chemicals used were of analytical grade, and all solvents were of HPLC grade. Fully O-sulfonated glycosaminoglycans were prepared by chemical sulfonation according to the methods reported previously (28). Enzyme treatment. The assay for HAase activity was standardized to the turbidity reducing unit (TRU) with commercial hyaluronidase (Sigma type I-S hyaluronidase) as a standard. One TRU of hyaluronidase is dened as the amount of enzyme that decreases the turbidity-producing capacity of 0.2 mg HA to that of 0.1 mg HA in 30 min at 37C. The conditions for enzymatic digestion were as follows: Substrate solution (40 l of 0.1 mg/ml HA in 0.2 M sodium acetate buffer, pH 5.0) was mixed with 30 l of 0.2 M sodium acetate buffer (pH 5.0) and 10 l of a standard enzyme solution (0.0520 units) or a sample solution at 0C, and the mixture solution was incubated at 37C for 2 h. The enzyme action was terminated after a xed time by incubating the mixtures at 100C for 5 min. And then, 2 l of sample solution was analyzed using FIA. FIA system. A ow diagram describing the FIA system is shown in Fig. 1. The established conditions for the determination of the HAase activity by FIA were as follows: The FIA system was assembled with a pump (Jusco 880-PU, intelligent HPLC pump (Tokyo,

Japan), a variable sample injector (VMD-350; Shimamura Instrument Co., Tokyo, Japan), a double-plunger pump (PSU-2.5W, Shimamura Instrument Co.) for delivery of the reagents, a dry reaction bath (Type DB-3, Shimamura Instrument Co.), a uorescence spectrophotometer (Hitachi Model F-1050; Hitachi Seisakusho, Tokyo, Japan), and a chromatointegrator (D-2500, Hitachi Seisakusho). The ow rate of a carrier buffer solution was constant at 0.5 ml/min and sodium hydroxide (0.3 M) and 1% 2-cyanoacetamide were added to the carrier at a ow rate of 0.25 ml/min using a double-plunger pump. The mixture passed through a polytetrauoroethylene (PTFE) reaction coil (10 m 0.5 mm i.d.) set in a dry reaction bath thermostated at 110C and then through a PTFE cooling coil (2 m 0. 5 mm i.d.). The efuent was monitored by uorescence with excitation at 346 nm and emission at 410 nm. Human urinary hyaluronidase. Five hundred microliters of human urine was centrifuged and the supernatant was concentrated into 50 l by using a 30-kDa cutoff lter device (Microcon YM-30; Millipore Co., U.S.A.) at 4C. Both the protein concentration and the hyaluronidase activity of the urine concentrate were determined. Protein concentration was measured by colorimetry with the Wako Urinapy Protein Kit (Wako Pure Chemicals Inc., Osaka, Japan) according to the manufacturers instructions. 1 H NMR spectroscopy. 1H NMR spectroscopy was performed using the conditions described previously (29). Briey, glycosaminoglycan, oversulfated glycosaminoglycan, or glycosaminoglycan-derived oligosacchaides (approx 2 mg) was dissolved in 0.5 ml of D 2O (99.9%) and freeze-dried repeatedly to remove exchangeable protons. The sample was kept in a desiccator over phosphorus pentoxide in vacuo overnight at room temperature. The thoroughly dried sample was then dissolved in 0.5 ml of D 2O (99.96%) and passed through a 0.45- m syringe lter and transferred to a NMR tube (5.0 mm o.d. 25 cm, pp-528; Wilmad Glass Co., Buena, NJ). 1D NMR experiments were performed on a JEOL GSX500A spectrometer equipped with a 5-mm eld gradient tunable probe with standard JEOL software at 303 K for NOE spectra or 333 K for other experiments on 500- l samples. The HOD signal was suppressed by presaturation during 3 s for 1D experiments.

TABLE I

Comparison of Fluorogenic Reagents for the FIA of Hyaluronidase Activity

Reagent 2-Cyanoacetamide Guanidine Arginine Ethylenediamine Taurine HA HAase a 40.0 18.0 0.32 0.16 0.04 HA a 1.0 1.7 0.1 0.04 0.02 Ratio 40 11 5.3 4.0 2.0

a 500 g/ml HA was incubated with and without 1 U/ml HAase for 3 h at 37C.

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TOIDA ET AL. kee, WI) at 85% purity. The oligosaccharides were labeled at their reducing ends with the AGA uorophore by reductive amination according to the procedure of Park et al. (29). AGA was used only after repeated recrystallization from deionized water (30). The CE experiments were performed on a Beckman capillary electrophoresis system P/ACE 5010 (Fullerton, CA) equipped with an ultraviolet detector. System operation and data handling were fully controlled using version 0.4 P/ACE station on an IBM-compatible PC. The CE system was operated in the reverse-polarity mode by applying the sample at the cathode and running using 20 mM phosphoric acid adjusted to pH 3.5 with 1 M dibasic sodium phosphate as previously described (29, 31). The capillary (75 m i.d., 375 m o.d., 57 cm long) was automatically washed before use with 0.1 M sodium hydroxide, followed by distilled water and then running buffer. Samples were applied using nitrogen gas pressure injection (5 s), resulting in a sample volume of 8 nl. Each experiment was conducted at a constant 15,000 V. Data collection was at 254 nm.

RESULTS

Optimization of the FIA Method

FIG. 2. FIA proles for the determination of HAase activity. Assay conditions and FIA conditions are described in the text. (Inset) Reproducibility of the FIA method is shown.

Capillary electrophoresis. To conrm the products due to the action of HAase digestion by capillary electrophoresis, the oligosaccharide products were labeled by monopotassium salt 7-amino-1,3naphthalenedisulfonic acid (AGA), which was from Aldrich (Milwau-

There are many reports on the determination of carbohydrates by postcolumn HPLC using uorometric derivatization (for review, see 32). We tested a number of the reported derivatizing reagents, including arginine, guanidine, ethylenediamine, taurine, benzamidine, and 2-cyanoacetamide, under optimized conditions. Table I shows the comparison of the relative intensities obtained from the reaction of each reagent

FIG. 3. CE analysis of the digestion mixture after derivatization with monopotassium salt 7-amino-1,3-naphthalenedisulfonic acid (AGA). CE analysis was performed on the reverse polality. The sample contained 400 mTRU HAase and was treated under the conditions as described in the text.

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FIG. 4. One-dimensional NMR spectra of the major oligosaccharides obtained under the conditions of hyaluronidase digestion. (Signals) 1, H-1 of GlcNAc at the reducing end; 2, H-1 of GlcNAc at the reducing end; 3, H-1 of the internal GlcNAc residues; 4, H-1 of GlcA attached to GlcNAc at the reducing end; 5, H-1 of the internal GlcA residues; 6, H-2 of GlcNAc at the reducing end; 7, H-6 of the internal GlcNAc residues; 8, H-2 of the internal GlcNAc residues; 9, H-4 and H-5 of the internal GlcNAc residues; 10, H-3 of the internal GlcA residues; 11, H-3 of the internal GlcNAc residues; 12, H-2 of the internal GlcA residues; 13, H-2 of GlcA at the nonreducing end.

with enzymatically treated HA as described under Materials and Methods. Based on the ratio of the high uorometric response of the products against that of the background, 2-cyanoacetamide was chosen as the uorogenic reagent for the FIA system. The optimum conditions for the postcolumn HPLC system using 2-cyanoacetamide had already been reported (32), and

these the reaction conditions (i.e., buffer pH, reaction temperature, and the length of reaction coil) were incorporated into the FIA method. Under the established conditions, the calibration curve for HAase activity was established as depicted in Fig. 2. The calibration curve, with peak heights as a function of activity, is not linear but rather a sigmoidal

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TABLE II

TOIDA ET AL.

Human Urinary Hyaluronidase Activity

Sample 1 2 3 4 5 6 Average SD mTRU 736 353 633 166 336 466 448 190 Protein (mg) 125 66 123 35 68 93 85 32 TRU/mg protein 59 53 51 46 49 50 51 4

curve (data not shown) in the range from 0 to 1 unit/ml. Samples can be diluted or concentrated to full within this calibration curve by suitable pretreatment procedures before enzyme digestion. Conrmation of the Products HA (10 mg) was treated with the standard enzyme (1 unit/ml) for 2 h and the products were tagged with

AGA according to the method described previously (29). As shown in Fig. 3, reaction mixture contains several oligosaccharides. To conrm the structure of each product, the reaction was scaled up 10 times and each oligosaccharide was puried by BioGel P-4 gel permeation chromatography for further identication by NMR spectroscopy. Five major oligosaccharides (assigned from 1 to 5 in Fig. 3) were puried by the preparative HPLC monitored at UV 205 nm. Figure 4 shows 1D NMR spectra of major fractions 1, 2, and 3 obtained from the digestion mixture. Based on the integration of each signal of the oligosaccharide, oligosaccharides 1, 2, and 3 were characterized as 4-, 6-, and 8-mers, respectively. The complete digestion of HA with testicular HAase produces tetra- and hexasaccharides. The partial degradation of HA under the conditions used in this paper procedes quantitatively affording additional higher oligosaccharides. By performing several assays on a solution of HA (0.1 mg/ml) it was estimated (means SD, n 6) that 0.1 mg of HA afforded about 1.8 0.2 nmol of reducing end groups (determined as GlcNAc), on HAase digestion. GPC

FIG. 5. Inhibition of unmodied and chemically O-sulfonated glycosaminoglycans on hyaluronidase activity. {, fully sulfonated GAGs; unmodied GAGs; A, chondroitin sulfate; B, dermatan sulfate; C, heparan sulfate; D, heparin; E, hyaluronan.

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analyses conrmed that in the absence of enzyme no additional reducing end groups were produced (data not shown). Human Urinary Hyaluronidase Activity A hyaluronidase activity of approximately 57 TRU/mg protein has been reported in human urine (33). The HAase activity measured in the urine of healthy human subjects by the present method (Table II) gave excellent agreement with the value reported by Csoka et al. (33). Inhibition of HAase Activity by Fully O-Sulfonated Glycosaminoglycans We previously reported that chemically fully O-sulfonated GAGs show high heparin cofactor II-mediated anti-coagulant activity (34). Using the HAase activity assay developed in the current study, the HAase inhibitory activity of these fully O-sulfonated GAGs was determined. The inhibitory effects of unmodied GAGs and chemicallyO-sulfonated GAGs on HAase are depicted in Fig. 5. All assays were performed in triplicate. Each of the O-sulfonated GAGs inhibited HAase in a dosedependent fashion, whereas unmodied GAGs (with the exception of heparin) did not show signicant inhibitory effects. The 50% inhibitory concentration (IC 50) of fully O-sulfonated GAGs on HAase was 1.35 g/ml (O-sulfonated chondroitin sulfate), 1.33 g/ml (O-sulfonated dermatan sulfate), 0.78 g/ml (fully Osulfonated hyaluronan), 1.28 g/ml (O-sulfonated heparan sulfate), and 1.14 g/ml (O-sulfonated heparin). The O-sulfonated hyaluronan was strongest among the sulfonated GAGs at inhibiting HAase. The IC 50 of porcine intestinal heparin chains inhibited HAase activity at the same level as fully O-sulfonated heparin. Figure 6 shows the LineweaverBurk plot of the inhibition of heparin and fully O-sulfonated HA on hyaluronidase. Heparin was found to inhibit hyaluronidase activity noncompetitively, while fully O-sulfonated HA was found to inhibit hyaluronidase through both competitive and noncompetitive effects.
DISCUSSION

FIG. 6. LineweaverBurk plot of inhibition of heparin and fully O-sulfonated HA on hyaluronidase. Each point represents the mean value of triplicate. HAase (50 TRU/mL) was used for the experiments. , untreated; F, heparin; E, fully O-sulfonated HA.

A new FIA method for determining HAase activity has been developed. The simple, specic, and highly sensitive uorometric detection made this assay useful as a screening method for measuring the inhibitory effects of sulfated polysaccharides such as heparin and chemically O-sulfonated glycosaminoglycans. The FIA method shows good reproducibility for the determination of the HAase activity compared with other previously reported methods.

HAase is also called spreading factor (35) and is a subject of intense interest in the cancer eld. Early studies presented contradictory results on the increase of HAase levels in tumors compared with normal tissues (36). Das and Chatterjee, using a turbidimetric assay, observed increased levels of HAase in metastatic vs. nonmetastatic tumors of different origins (ovary, breast, cervix, uteri, and lymph nodes) (37). Their conclusions were derived from pooled results because of low sample size, but no conclusion was reached for any given type of tumor. Recently, high levels of HAase have also been found in the urine of patients with nephroblastoma by Stern et al. (38), who suggested that this enzyme could be used as a new marker for this disease. Studies on prostatic cancer and breast cancer have shown that HAase activity was increased in malignant and metastatic tumors compared with normal tissue and primary breast tumor, respectively (39, 40). Based on these ndings, the FIA method may prove useful for the screening for the diagnosis of metastatic tumors. The inhibitory effects of unmodied GAGs and chemically O-sulfonated GAGs on HAase were also investigated by using the method established in this report. All of the O-sulfonated GAGs inhibited HAase in a dose-dependent fashion. Unmodied GAGs, with the exception of heparin, did not show strong inhibition. The O-sulfonated hyaluronan was strongest HAase inhibitor among the substances examined. Furthermore, the IC 50 of O-sulfonated hyaluronan is approximately three times higher than those of polyuronates such as pectic substances reported previously (1).

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TOIDA ET AL. 18. Hernandez, J. R., Garcia, J. M., Martinez, M. A., Allende, M. T., and Ruibal, M. A. (1995) Int. J. Biol. Markers 10, 149 155. 19. Natowicz, M. R., Short, M. P., Wang, Y., Dickersin, G. R., Gebhardt, M. C., Rosenthal, D. I., Sims, K. B., and Rosenberg, A. E. (1996) N. Engl. J. Med. 335, 1029 1033. 20. Koda, A., Nagai, H., Watanabe, S., Yanagihara, Y., and Sakamoto, K. (1976) J. Allergy Clin. Immunol. 57, 396 407. 21. Cox, J. S. G. (1967) Nature 216, 1328 1329. 22. Bonner, W. M., Jr., and Cantey, E. Y. (1966) Clin. Chim. Acta 13, 746 752. 23. De Salegui, M., Plonska, H., and Pigman, W. (1967) Arch. Biochem. Biophys. 121, 548 554. 24. Dorfman, A., and Ott, M. L. (1948) J. Biol. Chem. 172, 367. 25. Frost, G. I., Csoka, T. B., Wong, T., and Stern, R. (1997) Biochem. Biophys. Res. Commun. 236, 10 15. 26. Honda, S., Matsuda, Y., Takahashi, M., and Kakehi, K. (1980) Anal. Chem. 52, 1079 1082. 27. Toyoda, H., Motoki, M., Tanikawa, M., Shinomiya, K., Akiyama, H., and Imanari, T. (1991) J. Chromatogr. 565, 141148. 28. Maruyama, T., Toida, T., Imanari, T., Yu, G., and Linhardt, R. J. (1998) Carbohydr. Res. 306, 35 43. 29. Park, Y., Cho, S., and Linhardt, R. J. (1997) Biochim. Biophys. Acta 1337, 217226. 30. Lee, K. B., Al Hakim, A., Loganathan, D., and Linhardt, R. J. (1991) Carbohydr. Res. 214, 155168. 31. Toida, T., Yoshida, H., Toyoda, H., Koshiishi, I., Imanari, T., Hileman, R. E., Fromm, J. R., and Linhardt, R. J. (1997) Biochem. J. 322, 499 506. 32. Imanari, T., Toida, T., Koshiishi, I., and Toyoda, H. (1996) J. Chromatogr. 720, 275293. 33. Csoka, T. B., Frost, G. I., Wong, T., and Stern, R. (1997) FEBS Lett. 417, 307310. 34. Toida, T., Maruyama, T., Ogita, Suzuki, A., Toyoda, H., Imanari, T., and Linhardt, R. J. (1999) Int. J. Mol. Biol., in press. 35. Duran-Reynals, F.(1950) Ann. N.Y. Acad. Sci. 52, 946 957. 36. Cameron, E. (1966) in Hyaluronidase and Cancer, Pergamon, Oxford. 37. Das, S. K., and Chatterjee, P. C. (1981) J. Indian Med. Ass. 77, 2123. 38. Stern, M., Longkar, M. T., Adzick, N. S., Harrison, M. R., and Stern, R. (1991) J. Nat. Cancer Inst. 83, 1569 1674. 39. Lokeshwar, V. B., Lokeshwar, B. L., Pham, H. T., and Block, N. L. (1996) Cancer Res. 57, 778 783. 40. Bertrand, P., Girard, N., Duval, C., DAnjou, J., Menard, J-F., and Delpech, B. (1997) Int. J. Cancer 73, 327331.

These experimental results suggest that the FIA method might be useful to assay HAase activity linked to diseases and also to screen of the inhibitory effects of new substances on HAase activity of metastatic tumors.
ACKNOWLEDGMENTS
We are very grateful to Professor Robert J. Linhardt (The University of Iowa), who carefully read and improved the language of the manuscript. This work was supported in part by Grants-in-Aid from the Ministry of Culture, Sports and Education of Japan (09672185 (T.I.) and 09470490 (T.T.)) and the Nakatomi Foundation (T.T.).

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