Influence of human β-thromboglobulin on prostaglandin production by pig aortic endothelial cells in culture

THROMBOSISRESEARCH24; 95-103, 1981 0049-3848/81/190095-09$02.00/O Copyright (c) 1981 Pergamon Press Ltd.

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rights

reserved.

INFLUENCEOF HIJMN B-THROMBOGLOBULIN ON PROSTAGLANDINPRODUCTIOY BY PIG AORTIC ENDOTHELIALCELLS IN CULTURE

A. A.R.C.

Institute

(Received

of

Ager

and J.L.

Gordon

Animal Physiology, CB2 4AT, U.K.

Babraham,

Cambridge

4.8.1981; in revised form 2.10.1981. Accepted by Editor V.V. Kakkar)

ABSTRACT One batch of human E-thromboglobulin (RTG) was found to inhibit PGIz production by pig aortic endothelial cells in culture. PGE2 production by these cells was also inhibited. BTG had a similar inhibitory effect on prostaglandin production by pig aortic smooth muscle and mouse 3T3 cells and this inhibitory activity was reduced Three subsequent to 50% of control value on Amicon filter dialysis. batches of BTG were found to bc without effect on prostaglandin production by aortic endothelium and 3T3 cells. Ammonium acetate and sodium azide, which are used during the preparation of BTG, were tested for inhibitory activity and were active against endothelial cell prostaglandin production only at concentrations that were cytotoxic; these ions were therefore unlikely to account for the inhibitory activity of ETG initially seen. Freshly isolated endothelial cells and subcultured endothelial and smooth muscle cells at different stages of growth to confluence were also tested to see if the previously described ETG receptor had been lost during growth of cells in culture. The effect of yTG or low affinity platelet factor 4 (LA-PF4), the precursor of 8TG, was compared with that of BTG. No inhibitory effects of ETG or yTG were found under these conditions. We therefore conclude firstly, that any effects of ETG or yTG on prostaglandin production by vascular cells in culture cannot be reproducibly demonstrated, .and secondly, that any such effect is not specific for PGIz or for endothelium.

INTRODUCTION B-thromboglobulin (BTG) is a platelet specific protein which is released from the a-granules during platelet aggregation in response to a variety of stimuli (1, 2). It is the most abundant protein secreted during the release reaction. The biological function of ETG remains unknown; although it this is unlikely to be its major biological function binds to heparin, from a-granules when because platelet factor 4, which is also released platelets aggregate, has a much higher affinity for heparin than ETG. 95

‘o.,, Hoi~e et a; (3j tnat h:133~. _ ‘7; ‘:in,?s sFe;ifisallv Ii, to _ A: prostalyclin (PCI~) production by :::itured bo:inr aortic endotheiial cells led these xorkers to propose ti;at ZTG’s function is to promote piatelet aggregation b>- reducing l;ascl;lar xsll PC12 production, thus remol:ing the inhibitory effect of PGI? on ylsrelet $oposition or on platelet aggregat ion . However , it was not clear from the e.xoeriments of Hope et al Khether the inhibitor); effect of 3TG ~3s exerted specificall> on endothelium and specifically on PC12 production. PGI2 is produced not only bv vascular endothelium but also bv the subjacent smooth muscle cells of the’vascular vail (-1 ;, 6) , and both these cell types in culture also produce PGEz (7, .s, 9),‘uilich can antagonise the in!libitory effects of FGIz (10, 11). KC therefore measured the effects of several batches of 3X on the production of PGIz and FGEz by pig aortic endothelium, pig aortic sn;ooth muscle and mouse 3T3 cells in culture. Xe also tested a sample of *fTG (lo\<-affinity platelet factor 4; LA-PF:) , :ihich is the immediate precursor of STG, having four additional amino a
discoveT...-

and inhibits

MATERIALS AND MET~IODS Cell

Culture

Pig aortic cndothelial and smooth muscle cells were cultured as previously described (13, 14). BALB/c 3T3 cells, clone A31 were purchased from Flop Laboratories (Irvine, Ayrshire, U.K.) and grown as for smooth muscle cells. For experiments with BTG, cells were grohn in 16 mm diameter wells of tissue culture plates (Falcon 3008, Becton-Dickinson (UK) Ltd., Wembley The growth medium Pliddx, UK) and used when confluent (s lo5 cells/well). was removed, the cells were rinsed once with Ca2+ and Mg2+-free phosphate buffered saline (Dulbccco ‘A’ tablets, Oxoid Ltd., Basingstoke, Hants, UK) The and fresh medium containing BTG in the range 1 - 100 pg/ml was added. cells were then incubated either for 3 h or overnight (“- 18 h) at 37°C and Foetal calf the supernatants collected for prostaglandin radioimmunoassay. serum was added to the medium during overnight incubations (10% v/v for The amounts 20% v/v for endothelial cells). smooth muscle and 3T3 cells; of 6-keto-PGFlo (the stable non-enzymic breakdown product of PGI2) and of PGE2 in the culture medium were measured by radioimmunoassay as previously described (8). Human BTG, prepared according to the method of Moore et al (1 ), was kindly supplied by Dr D.S. Pepper, Blood Transfusion Centre, Edinburgh. It was stored frozen at 1 mg/ml in 154 ml\1saline and diluted in growth medium immediately before use. Four different batches were tested and they are Two preparations of unpurified referred to as Batch 1 - 4 in the text. platelet release products were also tested. These preparations had not been subjected to the preparative procedure used for BTG and therefore it Although 3TG is is unlikely that hydrolysis of yTG to BTG had occurred. the major protein released from aggregating platelets these preparations also contained other platelet proteins and granule constituents as well as A23187). yTG the agents used to induce secretion (thrombin or ionophore

Vo1.2&, h'o.1/2

PROSTAGLAXDI?jPRODUCTION

was kindly supplied by Dr C.N. Chesterman, Uni;-ersityof Melbourne, Department of Yedicine. RESULTS 1.

Inhibition of urostaelandin oroduction bv 'TG

Preliminary experiments established that human 3TG (Batch 1) inhibited the production of PGIa by pig aortic endothelial cells in culture after overnight incubations; the dose giving 50% inhibition was 20 gg/ml. Production of PGEz by endothelial cells was also inhibited by STC. Subcultures of pig aortic smooth muscle cells produced mainly PGE2 and the inhibitory effect of $TG on smooth muscle cell PGEz production was similar to its effect on endothelial cells. These results are summarised in Fig. 1. z

FIG. 1 Inhibition of prostaglandin production by human B-thromboglobulin (0) 6keto-PGFio and (A) PGE2 production by endothelium; (A) PGE;!by smooth muscle. Error bars represent S.E.M.

When comparable experiments were performed with 3T3 cells (which produce only PGEa) ETG exerted a similar inhibitory effect; control value, 4.0 + 0.2 ng PGEa/well; + 10 ug/ml BTG, 3.3 + 0.4 ng PGEa/well; + 30 ug/ml BTG 2.1 + 0.2 ng PGEa/well (n = 4). Similar-inhibitory effects were observed-if BTG was in contact with the cells for only 3 hours; for example prostaglandin production by subcultured endothelium was reduced from 0.95 + 0.01 ng 6-keto-PGFro/well to O.Si 2 0.01 ng 6-keto-PGFlo at 30 ug/ml BTG and to 0.45 + 0.01 ng 6-keto-PGFlo at 100 ng/ml ETC. When this batch of BTG was dialysed in an Amicon, with a 15,000 M. Wt. cut-off, its inhibitory effect was reduced (see Table 1).

97

98

?ROST.AGLMDLU ?RODLCTIO?;

Vol.‘&,

40.1/z

TABLE 1 Effect

of

dialysis

of

uroduction

6TG on the

by cultured

inhibition

endotheliat

of ceils

Non-dialysed

10 ugiml BTG

1Sh

-‘h

1Sh

134 2 2

97 5 -1

97 i S

s7 + 10

;?g/ml STG

Results 2.

Effects

of

58 2 1

33 ? 2

110 z 6

7s + 6

47 ? 1

20 i 3

s7 r 3

50 ? 7

expressed

different

Dialysed

-2h

30 :g/ml 3TG 100

FGIz

as percentage

batches

of

of

control

r S.E.bl.

in = 4)

@TG

The effects of four different batches of STG on prostaglandin production by pig aortic endothelium and mouse 3T3 cells in culture summarised in Table 2; batch 1 was active, but batches 2-4 had no inhibitory effects on prostaglandin production.

are

TABLE 2 Effect

of

four

different

endothelial

cells

batches

of

BTG on PGI;! production

and PGE2 production Endothelial *sf3

by 3T3 cells

cells

ST3 cells

1

2

Batch

I

Batch

2

Batch

3

99 + 12 (11)

Batch

4

13s + 17 (11)

*s2

(4) 93

172 ? 39 (3)

(6)

14s + 6 (3) 113 A 16 (4)

* Tested at 30 ug/ml. Results expressed as percentage of control C S.E.M. in 3h incubations. No. of observations in brackets.

_

3.

Effects

of

Because

ions

used

in the

preparation

ammonium and acetate

+ 3

(4) 4

!:

by

ions

of are

BTG tested

at

50 ug/ml

BTG used

in

the

preparation

of

vo1.24,

PROSTAGLMDI?i PRODCCTION

x0.1/2

99

BTG and sodium azide is present as a preservative, the effects of these ions (which can be carried through the preparative procedure and concentrated during lyophilisation) were tested on prostaglandin production. Table 3 shows the effects of ammonium acetate and sodium azide on prostaglandin production by endothelium and 3T3 cells during 3h incubations. Sodium azide up to 0.1% (?J 15 mM) had no inhibitory effect on either cell type. At higher concentrations prostaglandin production was inhibited in both cell types but 1% sodium azide was cytotoxic as assessed by microscopic observation (cells started to round up and pull away from substrate).

TABLE 3 Effect

of

ions

used

in the

production

by endothelial

Endothelial Azide

Results n = 4.

preparation

of

BTG on prostaglandin

and 3T3 cells 3T3 cells

cells Acetate

Azide

Acetate

0.03%

124 r 4

96 ? 7

93 2 3

95 t 4

0.1%

111 ? 3

82 + 2

130 !: 13

130 k 19

0.3%

76 2 2

65 ? 2

42 ? 5

153 ? 12

1.0%

26 2 5

45 +

a

24 + 2

240 + 36

expressed

as percentage

of

control

? S.E.M.

during

3h incubations.

Ammonium acetate up to 0.1% (12 mkl) was also without significant effect. At higher concentrations endothelial cell PGIz production was inhibited whereas 3T3 cell PGE2 production was stimulated, and again 1% ammonium acetate was cytotoxic as assessed by microscopic observation,on both cell types .

During overnight incubations sodium azide inhibited PGIz production by endothelium but not in an obviously dose dependent manner; production was 40 t 1% of control value at 0.01% (n = 4) ; 79 + 12% at 0.1% (n = 4) and 55 + 4% at 0.3% (n = 4). Overnight incubation with ammonium acetate again showed a differential effect on prostaglandin production by endoPGIz production by endothelium was not affected up thelium and 3T3 cells: to 0.3% ammonium acetate (112 ? 2% of control (n = 4)) whereas PGEz production by 3T3 cells was stimulated to 204 k 14% of control (n = 4) at this concentration.

4.

Effects different

of

BTG and yTG on prostaglandin stages

of

production

by cell

cultures

growth

Experiments with endothelial and smooth muscle cells performed on subcultured cells (up to 16 passages) shortly

were routinely after reaching

in

iG0

PROSTXGMDI?;

PRODcCTIc‘~;

:‘o;.zL,

so

confluence. It soened possible receptor for ZTG nig:ht that the nutati;.e be lost during successive subculture and \+e therefcrc! +est*d the effect ot‘ 3TG on prostaglandin production by confluent prizarl, - be an artefact of preparation and that -iTG is the physiological::: relesant molecule, we tested the effects of yTG under the same conditions. Neither 2TG (batch J! nor yTG at 50 ugjml inhibited the production of n->.ets-PGF:;i or or' PGE: (Table 4).

TABLE 4 Effect

of

8TG and yTG on prostaglandin

primarv

cultures

of

oroduction

endothelial

cells

6-keto-FGF,,

Results S.E.M.

are

by

PGEZ

Control

31 + 7 [3)

138 i

50 Fig/ml UTG

31 ?r 3 (3)

i52

SO ug/ml

39 lr 2 (3)

210 i 5 (3)

yTG

expressed

as pg of

prostaglandin/well

19 (3)

* ‘: (3)

during

2h incubations

r

To test the possibility that the effect of 3TG (or of -{TG) might be replicate experiments were performed on lost in post-confluent cultures, rapidly proliferating or confluent cultures of endothelial and smooth muscle cells. Neither ETG (batch 4) nor yTG at 50 gg/ml had any inhibitory effect on prostaglandin production by either cell type (Table 5).

TABLE 5 Effect

of

$TG and yTG on prostaglandin

rapidly

growing Endothelial

and confluent cells

production cell

by

cultures

Smooth muscle Rapidly growing

cells

Rapidly growing

Confluent

0.16 ?: 0.02

0.15 + 0.02

i 0.02

1.19 ? 0.11

ug/ml BTG

0.14 2 0.01

0.23 f 0.03

0.69 * 0.04

1.24 + 0.10

50 ug/ml YTG

0.18 ? 0.02

0.23 i 0.02

1.29 i: 0.1s

1.40 + 0.45

Control 50

Results are mean values (ng of S.E.N. (n = 4) for prostaglandin PGFio measured for endotnelial

0.79

Confluent

prostaglandin/well during 3h incubations) t 6-ketoproduction by subcultured cells; PGE2 for smooth ~~s;ile cells. cells,

Vol,2S,

5.

so.112

Effect

of

PROST&XXUDIN PRODUCTION

unpurified

platelet

release

101

products

The effects of two preparations of unpurified platelet release products on prostaglandin production by endothelial cells were tested; in one, release had been induced by thrombin (10Um:l)and in the other by ionophore A23187 ( 2 PM). At concentrations equivalent to 50 Fig/ml BTG both preparations stimulated PGIz production by confluent cultures of endothelium; during 3h incubations production was increased to 158 + 13.4%(n = 1S;C S.EJiJof control values by thrombin induced releasate and to 257.1 ? 32.5% (n = 3) by .A23187 induced releasate.

DISCUSSION The first batch of BTG that we tested was an effective inhibitor of PCIz synthesis by cultured pig aortic endothelial cells, but three subsequent batches had no effect. This discrepancy could have arisen because of variation in the cell culture conditions or because of variation in the batch was active on pig aortic preparation of BTG. However, the effective endothelial and smooth muscle cells and mouse 3T3 cells in culture whereas subsequent batches had no effect on any of these three cell types; one would therefore have to postulate that all cell types had simultaneously become unresponsive to BTG if a variation in cell culture were the explanation. The effective batch was equi-active on endothelial cell production of PGIz and PGEz, indicating that there was no specificity in its inhibitory effect towards PGIz production; our other experiments also demonstrated that there was no specificity with respect to cell type or species. This active batch of STG was effective when incubated with the cells in culture medium alone or when incubated overnight in culture medium with serum present. It is reasonable to presume that the bovine equivalent of BTG is present in high concentrations in the foetal calf serum used in these experiments - human serum contains lo-20 ug/ml BTG (2). Therefore, the equal effectiveness of this batch in the presence or absence of serum suggests that its inhibitory effect might not have been due to BTG per se; this conclusion is reinforced by our observation that the effectiveness of this batch was reduced after dialysis, which indicates that a low molecular weight component might be involved. We attempted to identify such a component by testing the effects of ions known to be present during the preparative procedure (e.g. ammonium acetate and azide). Although sodium azide and ammonium acetate did have inhibitory effects on prostaglandin production by cultured endothelium, these were only significant at high concentrations (> 120 mM), which were obviously cytotoxic as evidenced by morphological changes. Such cytotoxic effects were not observed on cells treated with the active BTG preparation, which suggests that none of the ions tested was responsible for the inhibitory effect 0% this batch. Evidence is accumulating that BTG is a proteolytic during preparation and that the major protein released we tested the effect of platelets is yTG (or LA-PF4);

artefact formed from stimulated yTG on prostaglandin

102

PROSTAGLiSDIX PRODL'CTIG?;

vo1.24, ?;3.1!1

production by endothelial and smooth muscle cells but found it tJ be ineffective. Ke also tested unpurified platelet release products, sne preparation that had been stimulated by thrombin and one by ionophore X3187; both preparations stimulated PGI: production by cultured endothelial cells. Although it is possible that the agents *used to induce platelet release might have been responsible for stimulating PGIz production, Coughlin et al, (16) shorted that purified piatelet derived growth factor (another protein secreted from a-granules during the platelet release reaction) increases PGI2 production by cultured bovine aortic endothelium, and xe and others (lS, 16) have previously shohn that serum (which contains all the platelet release products) also stimulated prostaglandin production by cultured end_otheliun; it therefore appears that the net effect of platelet release products may be to stimulate rather than to inhibit endothelial prostaglandin production. We considered the possibility that an effect of STG (or of -{TG)might not be easily demonstrable under the conditions routinely used for OUI experiments (subcultured cells tested shortly after reaching confluence) if the putative receptor were lost from post-confluent cells and/or during successive subculture. Accordingly, we tested ZTG (batch 1) and _iTC on primary cultures of endothelium and on preconfluent subcultures of endothelial and smooth muscle cells that were proliferating rapidly. Ke observed no inhibition of prostaglandin production under any of these circumstances, which indicated that the ineffectiveness of ZTC and ‘/TG was not a consequence of the particular conditions chosen for our routine experiments . In conclusion we were unable to show a reproducible effect of _TG or yTG on prostaglandin production by endothelial cells or other cell types in culture, although the one batch of BTG that was effective was equiactive on endothelium, smooth muscle and 3T3 cells, showed no selectivity in its inhibitory effect on PC12 production compared with the production of PGEz, was not affected by the presence of serum, but was rendered less active after dialysis. These results cast some doubt on the role of BTG as an inhibitor of prostaglandin production, as does the observation that unpurified platelet release products stimulate prostaglandin production. At the very least, our results show that any effect of BTG or yTG on prostaglandin production cannot be consistently demonstrated, and therefore any investigator contemplating a study of this subject should be aware of this. REFERENCES 1.

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2.

MOORE, S. a platelet Pathology, Biomedical

_

HOPE, W., MARTIN, T.J., CHESTER&W, C.N. and blORGAV, F.J. Human !3-thromboglobulin inhibits PGI2 production and binds to a specific site in bovine aortic endothelial cells. Nature 2SZ I _710-213, 1979. --~ __._ ____

3.

and PEPPER, D.S. Identification specific release product. In: J. L. Gordon (Ed.) Amsterdam: 1976, pp. 293-311. Press,

and characterisation of Platelets in Biology-a& Eisevier/North_and

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Differential MONCADA, S., HE&&v, X.G., HIGGS, E.A. and VANE, J.R. formation of prostacyclin (PGX or PG12) by layers of the arterial wall. An explanation for the anti-thrombotic properties of vascular endothelium. Thromb. Res. 11, 323-34-l,1977.

5.

Effect of endothelial damage on prostagPURi, and NEEDLEMAN, P. glandin synthesis by isolated perfused rabbit mesenteric vasculature. J. Cardiovasc. Pharmaco_l_. A, 299-309, 1979. --_

6.

BUCHXN&N, M.R., DEJANA, E., CAZENAVE, J.-P., RICHARDSON, >I., MUSTARD, J.R. and HIRSCH, J. Differences in inhibition of PGIz production by aspirin in rabbit artery and vein segments. Thromb. -Res. 2, 437-460, 1980.

7.

ALEXAINDER,R.W. and GIMBRONE, M.A. Stimulation of prostaglandin E synthesis in cultured human umbilical vein smooth muscle cells. Proc. Natl. Acad. Sci. -- USA 73, 1617-1620, 1976. _---_

8.

AGER, A., GORDON, J.L., MONCADA, S., PEARSON, J.D., SALMON, .J.A.and TREVETHICK, M.A. Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells. J_. Cell.~__. Physiol. In press. .-

9.

ODY, C.! SILLAN, C. and RUSSO-MARIE, F. Identification of the prostaglandins produced by cells in culture from the three layers of the pig aortic wall. -Acta Therapeutica 6, Suppl. 4, 55, 1980. --

10.

OMINI, C., MONCADA, S. and VANE, J.R. The effects of prostacyclin (PGI2) on tissues which detect prostaglandins. Prostaglandins 12, 625-632, 1977.

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GORDON,

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NIEWIAROWSKI, S., WALZ, D.A., JAMES, P., RUCINSKI, B. and KUEPPERS, F. Identification and separation of secreted platelet proteins by isoelectric focusing. Evidence that low affinity platelet factor 4 is converted to &thromboglobulin by limited proteolysis. ___ Blood s, 453-456, 1980.

13.

Lipid metabolism in cultured aortic smooth muscle PEARSON, J.D. Atherosclerosis 24 233cells and comparison with other cell types. ..-------._. .~ _ __’ 242, 1976.

14.

PEARSON, J.D., CARLETON, J.S., HUTCHINGS, A. and GORDON, J.L. Uptake and metabolism of adenosine by pig aortic endothelial and smooth muscle cells in culture. _-.__ Biochem. J. 170 .--J 265-271, 1978.

15.

Localisation and MACINTYRE, D.E., PEARSON, J.D. and GORDON, J.L. Nature 271 stimulation of prostacyclin production in vascular cells. ---' 549-551, 1978.

16.

J.L., PEARSON, J.D. and MACINTYRE, D.E. Effect of prostaglandin EZ on prostacyclin production by endothelial cells. Nature 278, 180, 1979.

COUGHLIN, S.R., MOSKOWITZ, M.A., ZETTER, B.R., ANTONIADES, H.N. and LEVINE, L. Platelet-dependent stimulation of prostacyclin synthesis by platelet derived growth factor. -. Nature 288, 600-602, 1980. ._ --