Heparin stimulates the release of glycosaminoglycans from cultured human endothelial cells

THROMBOSIS RESEARCH 57; 163-168, 1990 0049-3848/90 $3.00 t .OO Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

COMMUNICATION

BRIEF

HEPARIN STIMULATES THE RELEASE OF GLYCOSAMINOGLYCANS CULTURED HUMANENDOTHELIAL CELLS

Toshiyuki Department Toyama

Kaji

and

Nobuo

FROM

Sakuragawa

of Clinical Laboratory Medicine, Faculty of Medicine, Medical and Pharmaceutical University, 2630 Sugitani, Toyama-shi, Toyama 930-01, Japan (Received 6.9.1989; accepted in revised form 25.10.1989 by Editor W.H. Seegers)

INTRODUCTION and sulfated molecule, has been used Hepari n , ah igh an ionic acts by an anticoagulant drug. Heparin clinically as involved inhibition of serine proteinases anti thromb i n-med iated However, the antithrombotic properties of in blood coagulation. al ways quantitatively heparin and its fractions in vivo are not correlated neither with their anticoagulant potency in vitro nor for anti thrombin m (1). been with their affinity It has interacting capable of proposed that heparin is with blood vessels. Heparin binds to endothelium (2) and may exhibit the anticoagulant action on the surface. We hypothesized that heparin facilitate the release of endogenous glycosaminoglycans such may as heparan sulfate of endothelial cells, which is capable of producing The present study was unantithrombotic effect (3). dertaken to clarify that heparin can stimulate the release of glycosaminoglycans from cultured endothelial cells.

The 24- and 6-well -_-_-------Key words : Endothelial sulfate,

culture cells, heparin.

plates

coated

glycosaminoglycans,

163

with

gelatin heparan

were

ob-

164

HEPARIN AND ENDOTHELIAL CELLS...

Vol. 57, No. 1

tained from Corning (NY, U.S.A.). RPM11640 medium was purchased from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan). Fetal bovine serum was from Filtron (Australia). Na2 [35Slo4 (788 mCi/mmol) was purchased from New Eng 1 and Nuclear Corp. (MA, U.S.A.). Chondroi tin ABC lyase, chondroi tin 4-sulfate, chondroi tin 6sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid were from Seikagaku Kogyo Co.,Ltd. (Tokyo, Japan). Dextran sulfate was purchased from Wako Purechemical Co. ,Ltd. (Osaka, Japan). Unfractionated heparin (UFH), human thrombin and endothelial cell growth supplements (ECGS) were obtained from Sigma Chemical Co. (MA, U.S.A.). Low molecular weight heparin (LMWH) was from Kabivitrum (Sweden). Cell Endothelial cells were isolated from ._..__ .__.c.uLLtur &__ .R~o_ced!Lre.. human umbilical vein according to previously descrived methods (4). The cells were cultured in RPM11640 medium supplemented with 20% fetal bovine serum and 200 ,u g/ml ECGS in 5% CO2 in air at 37 “C . T r .e..-._. a t .me The cells were cultured until confluent using 24-... _.._n.t_& well culture plates. The cell density was approximately 4x 105 cells/cm2. The confluent cells were cultured in a fresh medium in the presence of 20 L1 Ci/ml C35Slsulfate (added as sodium sulfate) for 24 hr. After culture, the cell layer was washed 3 times with 11 mM HEPES buffer (pH 7.4) containing 137 mM NaCl, 4 mM KCl, 3 mM CaC12, 1 mM MgCl and 11 mM glucose and incubated in 0.25 ml of the buffer at 37 ? for 1 hr. An aqueous solution of UFH (1 USP U/ml or 10 fl g/ml), LMWH (1 anti-factor X a U/ml), chondroi tin 4-sulfate (10 ,u g/ml 1, chondroitin 6-sulfate (10 IL sulfate (10 II g/ml), g/ml 1, dermatan heparan sulfate (10 fi g/ml), hyaluronic acid (10 dextran sulfate (10 fi g/ml) or fi g/ml), thrombin (2 USP U/ml) was added to the incubation buffer.

A_n_a__lu~.l.cal..__..me.t.h-~.ds,. After

incubation, the buffer was harvested and the cell layer was washed with 0.25 ml of the fresh buffer. was mixed the harvested buffer. The total The wash with (35S-GAG) both in the t35S3sulfate-labeled glycosaminoglycans in the cell layer were measured by cetylbuffer and harvested pyridinium chloride precipitation (5). The 35S-GAG release (%) by deviding the radioactivity of 35S-GAG (dpm) caluculated was 35S-GAG released into the buffer by the radioactivity of (dpm) found in both the buffer and the cell layer. It is well known that the Ch~ar~ac.te_rLzaf lo??.. of .re!ea~.ed...35~S-GAG.. cells is endothel i al major component of glycosaminoglycans of an anticoagulant activity (3). sulfate which exhibits heparan ABC chondroitin Heparan sulfate is resistant to degradation by to that by nitrous acid characteristically sensitive lyase but (6). The 35S-GAG in both the harvested buffer and in cell the was treated with the enzyme (10 U/ml) or nitrous acid (240 1 ayer treatment mM in 10% acetic acid), and 35S-GAG resistant to each In the harvested precipitated by cetylpyridinium chloride. was resistant to about 75% of total released 35S-GAG was buffer, 35% was resistant to nitrous about ABC 1 yase and chondroitin about 80% of total 35S-GAG was resisIn the cell layer, acid. and about 25% was resistant to lyase chondroitin ABC tant to UFH the changed by were not These propor t ion nitrous acid.

Vol.

57,

No.

1

HEPARIN

AND ENDOTHELIAL

CELLS,.

.

165

Heparan sulfate release (%) was calculated by devidtreatment. radioactivity (dpm) of ing the chondroitin ABC lyase-resistant 35S-GAG in the harvested buffer by that found in both harvested Other glycosaminoglycans release (%I was buffer and cell layer. the nitrous acid-resistant same using calculated in the way radioactivi ty (dpm). Statistical nificance

by

Results t-test.

ana1ysi.s. Student’s

RESULTS

were

analyzed

for

statistical

sig-

AND DISCUSSION.

its fractions proposed that heparin and It has been may endogenous GAG which are capable of release of facilitate the In the present study, we producing antithrombotic effects (9). found that UFH significantly stimulated 35S-GAG release from culAs shown in Table 1, UFH (Fig. 1). tured endothel ial cells other GAG, sulfate and stimulated the release of both heparan about 75% of total released 35SIn this experiment, similarly. Since GAG was heparan sulfate in both control and UFH treatment. activity on the enheparan sulfate exhibits an anticoagulant we postulate that UFH-induced insurface (31, dothel ial cell crease in GAG release may contribute to the antithrombotic action the antithrombotic action of heparin of heparin. In other words, may be due to not only the antiserine protease actions but also a such as GAG release. stimulation of endothelial cell function (8) and tissue plasproteoglycan synthesis Heparin-stimulated have been minogen activator production (9) by endothelial cells reported.

IN THE ABSENCE OF THROMBIN

IN THE PRESENCE OF THROMBIN **

M"

20'

P al z '7 v) .z

10.

0.

CUNTRUL UFH LMWH

CONTROL UFH

FIG. 1. of 1 U/ml UFH and 1 U/ml Effect 35S-GAG (%I from cultured endothelial cell or presence of 2 U/ml thrombin. **Significantly control, P(O.01.

LMWH

LMWH on the release of layer in the absence different from

166

HEPARIN AND ENDOTHELIAL CELLS... TABLE

Vol. 57, No. 1

I

Characterization

of Released 35S-GAG (%I from Cultured dothelial Cells Treated with 1 U/ml UFH. __-_________-_______~~~~-~~-~~-~~_-~-~~--~_--_--_-~_-~__-__-_ Treatment Control -__-__-___________-_~~~~~~~-~~_-~_-~-~~-~~_-~_-~_-~_-~--~_-~_ Total

En

UFH treatment

8.58

+-

0.40

13.86

+

0.47***

8.21

+-

0.21

13.13

+

o.c5**

Othersb) 9.38 + 1.68 16.97 ______________________~~~~~_~~_~~_~~_~~~~~~~~~~~~~~~~~~~~~~~~

k

0.31*

Heparan

sulfatea)

a)35S-GAG resistant to chondroitin ABC lyase. b)35S-GAG resistant to nitrous acid. Values are means 2 SE of 4 samples. *Significantly Note. ferent from control, P
diffrom control,

10 II g/ml chondroitin 4-sulfate FIG. 2. Effect of 10 fig/ml UFH, 10 !J g/ml chondroitin 6-sulfate (C6S), 10 /J g/ml dermatan (C4S). (HS), 10 sulfate heparan II g/ml sulfate CDS), 10 fi g/ml and 10 fi g/ml dextran sulfate (DXS) on the hyaluronic acid (HYL), layer. ccl 1 endothelial cultured 35S-GAG from release of ***significantly **Significantly different from control, P
Vol. 57, No. 1

HEPARIN AND ENDOTHELIAL CELLS...

167

effect of several GAG and dextran sulfate on the _examine S GAG release from endothelial cells (Fig. 2). Dextran a synthetic polymer of sulfated glucose consisting of no sulfate, acids, as well as uranic UFH significantly increased 35S-GAG 4-sulfate, chondroi tin 6release. In contrast, chondroi tin sulfate, dermatan sulfate, heparan sulfate, and hyaluronic acid caused no significant change. These results suggest that sulfate are important to the groups rather than carboxyl ITroups UFHIn addition, LMWH did not induced increase in 35S-GAG release. show a stimulatory effect on 35S-GAG release as shown in Fig. 1. that to protein core, might bc We speculate 35S-GAG, 1 inked protein the directly and physicochemically displaced by UFH on 35S-GAG was increased by UFH treatsince the acid soluble core, shown). no t ment and the UFH stimulation occurred at 4 “c (data UFH would be an active site of the reaction, groups of Sulfate and a long sugar chain might contribute to reaching of UFH to the Thus, UFHlocation of the exchange of UFH for endogenous GAti. endothelial cell layermay partly from stimulated GAG release ccl 1 correspond to the concept of heparin binding to endothelial layer. the release of GAG linked to stimulates Al though thrombi n the mechanism is protein core from endothelial cell layer (lo), As UFH increased 35S-GAG release in the presence of clear. not of the UFH stimulation thrombin as shown in Fig. 1. the mechanism must be different from that of the thrombin stimulation. we found that UFH stimulated the release of enIn conclusion, The overall antithromdogenous GAG from endothelial cell layer. interaction bebotic action of heparin obviously includes -the Our findings will contribute to tween endothelium and heparin. from a viewpoint of enthe interpretation of heparin action dothelial cell function. theWs5

ACKNGWLEDGEMENT authors The assistance.

thank

Ms.

Naoko

Ejiri

for

her

fine

technical

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