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The use of human endothelial cells cultured in flat wells and on microcarrier beads to assess tissue plasminogen activator and factor VIII related antigen release
THROMBOSIS RESEARCH 41; 581-587, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
THE USE OF HUMAN ENDOTHELIAL CELLS CULTURED IN FLAT WELLS AND ON MICROCARRIER BEADS TO ASSESS TISSUE PLASMINOGEN ACTIVATOR AND FACTOR VIII RELATED ANTIGEN RELEASE
C.V. Prowse', N.R. Hunter', I.R. MacGregor', J.Dawed and D.S. Pepper' 'Scottish National Blood Transfusion Service Headquarters Unit Laboratory and %RC/SNBTS Blood Components Assay Group, 2 Forrest Road, and 'S-E Regional Blood Transfusion Centre. Royal Infirmary, Edinburgh, Scotland. M.M.
McArthur',
(Received 17.7.1985; Accepted in revised form 22.11.1985 by Editor M.J. Seghatchian)
INTRODUCTION Tissue plasminogen activator (t-PA) has been localised in vascular endothelium (l-3) and is released into the circulation following a variety of Since some of these stimuli also stimuli (reviewed by Prowse and Cash, 4). elevate t-PA in models such as perfused pig ear and rat hind limb (5,6) we were interested in investigating if cultured endothelium possesses the capacity to respond to agonists which are active in the whole organism or in isolated organs. Pilot studies on endothelial cells cultured in flat wells (7) indicated that the increased cell surface to medium volume ratio obtained with microcarrier cultures might be more suited to such experiments (8-10) and in this comunication we describe a comparison of the basal secretion of t-PA and factor VIII related antigen (FVIIIR:Ag) in flat well and microcarrier cell cultures and present preliminary data on agonist induced release.
MATERIALS AND METHODS Tissue culture ware was obtained from Costar, via Northumbria Biochemicals, UK. Cell culture media and supplements were from Flow Laboratories, Irvine, UK. Calcium ionophore A23187, bovine serum albumin (BSA) , crystal violet, trypan blue, cycloheximide, actinomycin D and &isopropyl fluorophosphate (DFP) were supplied by Sigma, Poole, UK. Triton X-100, glucose and citric acid were products of BDH, UK. Dimethylsulphoxide (DMSO) was obtained from Aldrich, UK. Cytoaex 3 microcarrier beads were purchased
Part of this work was presented Fibrinolysis, Venice, 1984. KEY WORDS:
at the 7th International
Congress on
Tissue plasminogen activator; factor VIII related antigen; human endothelial cell cultures; microcarrier beads; drugs.
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from Pharmacia, Sweden. Fibronectin was obtained from the Protein Fractionation Centre, Edinburgh, UK, and human serum from the Scottish National Blood Transfusion Service. Human c( thrombin was prepared as described previously (11). Endothelial Cell Culture: Endothelial cells were isolated from human umbilical cord veins (HUVEC) by the method of Jaffe et al (12) and cultured and characterised as described previously (13). HUVEC were grown to confluency on Cytodex 3 microcarrier beads by the method of Busch et al (9). Flat Well Cultures: Growth medium was removed from confluent endothelial cells cultured in 9.6 cm2 wells and, following two washes with Medium 199, modified (M 199), was replaced with 1 ml of conditioning medium (CM) consisting of M 199 containing 1 % w/v BSA plus any agonist or control vehicle to The wells were incubated at 37OC for 3,6 or 24 hr then the CM was be tested. removed, centrifuged for 6 min at 8,000 g and the supernatant stored at -40°C prior to radioimmunoassay (RIA) for t-PA and FVIIIR:Ag. The cell monolayer was washed and lysed with 0.5% w/v Triton X-100 in 0.01 M Tris-HCl, pH 8.2 then treated in the same way as the CM sample. Microcarrier Perfusion: 27ml aliquots of washed microcarrier beads covered by endothelial cells ( 1 10 cells/ml of packed beads) were placed in 0.7 cm diameter plastic chromatography columns containing 70 pm pore size filters (Amicon Wright, UK) and were perfused with CM at 0.2 ml/min at 37OC for PO min to obtain basal secretory values. Then agonists were added in a 1 ml pulse or continuously throughout the subsequent perfusion. 0.2 ml fractions of effluent were collected and assayed for t-PA antigen and FVIIIR:Ag. Microcarrier Incubation: Washed microcarrier beads covered by confluent endothelial cells were suspended in an equal volume of CM and 2 ml aliquots transferred to plastic tubes (Sarstedt, UK) where any agonist to be tested was already present in a volume of 50 ~1. The beads were maintained in suspension on a shaking table (Janke and Kunkel, W Germany) at 37OC and 0.5 ml bead-free CM was removed at various times with replenishment by fresh CM. Viability, was assessed by the ability to exclude trypan blue. CM and lysates were prepared and stored as for flat well cultures prior to assay. t-PA RIA: This was performed exactly as described previously, using 50 ~1 test samples in duplicate (14). The working range of the assay was l-80 ng/ml of test sample. FVIIIR:Ag RIA: The RIA was a conventional competition assay using a polyclonal antibody raised in rabbits and an immobilised second antibody separation system. FVIIIR:Ag was iodinated by the Iodo-Gen method to a specific activity of 400 KBq/pg. The assay had a working range of l-100 ng/ml of test sample based on a normal plasma pool content of 10 pg FVIIIR:Ag/ml. Miscellaneous: The cell count in microcarrier cultures was determined by counting nuclei in 0.1 M citric acid containing 0.1% w/v crystal violet and viability was checked by measuring the exclusion of a 2% w/v trypan blue solution (9).
RESULTS t-PA standard curves were constructed using assay buffer or CM (prior to conditioning) or lysing buffer as diluent and the three curves were
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Recovery experiments were performed in two ways. Firstly superimposable. purified t-PA was added at a range of concentrations to cell free CM which had been in contact with confluent HUVEC for 24 hr, and the CMs were assayed for Recoveries averaged 74% and were greater when the CM was t-PA antigen. When t-PA was inactivated by diluted prior to addition of purified t-PA. treatment with DFP before addition to CM the recoveries approached 100%. These results suggested.that CM contained components which modified t-PA to a form less readily recognised in the RIA and that the modification required a free active site. Secondly, when t-PA or DFP inactivated t-PA was added to confluent HUVEC and the resultant cell free CM was harvested and assayed after 24 hr an average recovery of 20% and 40% respectively was obtained. The low recovery of purified t-PA added to HWEC cultures appears partly due to adsorption on cell surfaces and partly to its modification to a form less well detected by the RIA. Whether t-PA secreted by HUVEC behaves in a similar fashion is presently being investigated. t-PA antigen accumulated in the CM of HWEC cultures in Flat Well Cultures: a time dependent fashion and was also detectable in the cell lysates. The expressed antigen was shown to be a synthetic product of the cells ince pre-5 treatment with cycloheximide at 2 pg/ml or actinomycin D at 4 x 10 M resulted in a marked reduction in both secreted and intracellular
120. t-PA antigen secreted
60
n 0
12 TIME (HOURS) FIG. 1
Relationship between passage number of HWEC line and secreted levels of t-PA
5 cell
??- ??= ~3; A- A= P6; ??- m= P8; 0 - o= P12; 0-o = P18 where P = passage number and each passage represents two population doublings
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concentrations of t-PA. In early passage cells (P3 and 4) lo-20 ng t-PA/lo' cells was typically secreted over a 24 hr period. The increase of secreted t-PA antigen as a function of time is shown in Fig.1. The rate of basal secretion was greater in late passage cultures with all cell lines examined. In all subsequent experiments control and treated cells were derived from the same cell line at the same passage number. The effect of thrombin at 1 u/ml At 3 hr after upon t-PA secretion was tested in five separate experiments. addition of thrombin secreted t-PA was 92 k 26% of the control value (3i2 S.D.) while at 24 hr it was 126 2 23% of the control valce.
treated 10 u/ml Calcium tration
t-PA secretion at 3,6 and 24 hr was similar in control and thrombincells while short-term secretion of FVIIIR:Ag was stimulated by51 and thrombin and also at the early times by calcium ionophore (10 M) . ionophore reduced both t-PA secretion and the intracellular concenof t-PA over the 24 hr period by approximately 80%.
Microcarrier Perfusion: Microcarrier beads covered by confluent HUVEC were t-PA release over the first 60 min perfused over 5 hr with CM at 0.2 ml/min. was erratic, but after this time a steadier rate of secretion was observed. The rate of this steadier basal secretion of t-PA antigen in 15 perfusion experimgnts with 3 cell lines all at passages between 5 and 9 was 30 ?r 27 pg t-PA/10 cells/min (mean k SD). This was quantitatively similar6to the typical rate of secretion in flat well cultures of 42 pg t-PA/10 cells/min, but because of the high cell surface to medium volume ratio obtainable in microcarrier perfusion, this system can be used to detect short-term changes in t-PA concentration.
-30
0.1 t-PA antigen secretec I
FVlll R : Ag * m‘? secretea LU (ng /lObcells
c
( ng /106cells)
.lO
0
I-
0
,O
8
20
40
60
80
TIME lMlN 1
FIG. 2 Secretion of t-PA and FVIIIR:Ag by perfused HUVEC attached _5 to microcarrier beads. M. Effects of calcium ionophore A23178 at 10 Time 0 follows a 60 min equilibrating perfusion period. t-PA,
O-0;
FVIIIR:Ag,
.---..
Hatched bars indicate presence of ionophore
1
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ENDOTHELIAL CELL t-PA
585
Basal secretion of FVIIIR:Ag was also detectable in the microcarrier In six expegiments using HWEC at passage 5 the basal perfusion system. secretion was 13.0 f 12.1 ng/lO cells/min (mean f SD) and E'VIIIR:Ag would be undetectable over such short time courses in flat well cultures. Figure 2 shows basal secretion of t-PA and E'VIIIR:Ag by perfused cells on microcarriers and the transient stimulation of F'VIIIR:Ag release by calcium ionophore which was seen in each of three separate experiments. Because of the variation in basal secretion during Microcarrier incubation: perfusion experiments a time course of basal synthesis and secretion of t-PA and FVIIIR:Ag was set up over O-180 min using a discontinuous batch perfusion system and both antigens were secreted in approximately a linear fashion with respect to time although the earliest (5 min) sample invariably contained more antigen than expected from the secretion versus time curve. The amount of t-PA and6E'VIIIR:Ag typically secgeted by an early passage cell line was 15 pg/lO cells/min and 14 ng/lO cells/min respectively. Calcium ionophore stimulated F'VIIIR:Ag release at 10 min (157% of control 10 min value) while t-PA secretion was inhibited.
DISCUSSION By using an RIA we have been able to quantify t-PA antigen produced by cultured human endothelium in conditions where no biological activity can be detected because of the presence of excess plasminogen activator inhibitor (15-17). The t-PA was shown to be a synthetic product by using protein synthesis inhibitors. Recovery experiments indicated that t-PA with a free active site forms inhibitor complexes less readily detectable in the RIA as reported by Rijken et al (18) although the immunoassay of Levin et al detected both free and inhibited t-PA equally well (19). Recovery of added t-PA from cell cultures suggested that t-PA may be adsorbed to cell surfaces, and if this also occurred with native t-PA an underestimate of secretory rates would result. Further experiments are needed to establish the disposition of t-PA after secretion by cultured HUVEC. We observed differences in basal secretion of t-PA between cell lines and an increase with gigher passage number. In early passage cells our values of lo-20 ng/lO cells/24 hr corn qred with published values for primary or first passage HUVEC of about 5 ng/lO /24 hr (18,19). Alterations in expression of endothelial metabolites following repeated subcultivation has been reported for angiotensin converting enzyme and prostacyclin (which decrease) while 5'-nucleotidase increases (20-22). Conversely the secretion of some metabolites such as thrombospondin are unaffected by serial subcultivation (13). The reason for induction of t-PA synthesis and secretion in late passage cells is unknown but merits further investigation as a model of occurrences in vivo in areas of high endothelial turnover during chronic endothelial injury. Nor is it known whether concomitant changes in the synthesis of t-PA inhibitors occur. We have shown here that HUVEC cultured on microcarrier beads secrete t-PA and F'VIIIR:Ag at confluence at similar rates to HUVEC grown on conventional tissue culture plastic surfaces, but because of the former's much greater cell surface to medium volume ratio we could detect the release of these proteins over a considerably shorter interval showing that the microcarrier cultures are theoretically suitable for measuring short term release reactions. While calcium ionophore was shown to promote rapid release of
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FVIIIR:Ag in the perfused microcarrier cell system we have been unable to demonstrate release of t-PA by a range of agonists (including DDAVP, platelet activating factor and bradykinin) which have been shown by others to promote its release in intact animals and isolated organs (unpublished observations). Other agents which increase t-PA secretion may do so by effects upon Thus thrombin in the presence of serum induced t-PA protein synthesis. release from HUVEC with a 6 hour lag and a maximum effect at 16 hours (19) and Therefore, the such studies are more suited to flat well culture systems. use of flat well and microcarrier ccl 1 cultures should be seen as complementary for investigating modulators of t-PA secretion.
ACKNOWLEDGEMENTS Purified We thank Miss S. Maguire for expert technical assistance. t-PA was kindly provided by Dr. D. Collen, Leuven, Belgium, and Dr. M. We acknowledge the support of the Medical Einarsson, Kabi Vitrum, Sweden. Research Council (Project Grant G8217210SB) to C.V.P. and I.R. MacG., (Programme Grant ~G8218470) to J.D. and D.S.P. and the British Heart Foundation (BHF Grant 82/13) to I.R.MacG. and D.S.P.
REFERENCES 1.
TODD, A.S., The histological Pathol. 78, 281-283, 1959.
localisation
of fibrinolysin
activator.
2.
RIJKEN, D.S., WIJNGAARDS, G. and WELBERGEN, J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb. Res. I-&, 815-830, 1980.
3.
HOLMBERG, L., KRISTOFFERSON, A.C., LECANDER, L., WALLEN, P. and ASTEDT, B. Immunoradiometric quantification of tissue plasminogen activator secreted by fetal organs. Stand. J. Clin. Lab. Invest. 43, 347-354, 1982.
4.
PROWSE, C.V. and CASH, J.D. Physiologic and pharmacologic Sem. Thromb. Haemostas. 2, 51-60, 1984. fibrinolysis.
5.
MARKWARDT, F. and KLOCKING, K.P. Studies on the release of plasminogen activators. Thromb. Res. S, 217-223, 1976.
6.
EMEIS, J.J. Perfused rat hind legs. A model to study plasminogen activator release. Thromb. Res. 30, 195-203, 1983.
7.
HUNTER, N.R., MacGREGOR, I.R., McARTHUR, M.M., TAYLOR, P.M., PROWSE, C.V. and PEPPER, D.S. Tissue plasminogen activator measured by radioimmunoassay in human endothelial cell cultures. In: Progress in Fibrinolysis. J. Davidson, (Ed). Churchill Livingstone 1984, 1, (in press).
8.
PEARSON, J.D., CARLETON, J.S. and HUTCHINGS, A. Prostacyclin release stimulated by thrombin or bradykinin in porcine endothelial cells cultured from aorta and umbilical vein. Thromb. Res. 2, 115-124, 1983.
enhancement
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BUSCH, C., CONCILLA, P.A., de BAULT, C.E., GOLDSMITH, J.C. and OWEN, W.G. Methods in laboratory investigation - use of endothelium cultured on microcarriers as a model for the microcirculation. Lab. Invest. 47, 498-504, 1982.
10.
Superfusion of rat anterior pituitary cells SMITH, M.A. and VALE, W.W. Endocrinology, attached to Cytodex beads: validation of a technique. 107, 1425-1431, 1980.
11.
DAWES, J., JAMES, K., MICKLEM, L.R., PEPPER, D.S. and PROWSE, C.V. Monoclonal antibodies directed against human a-thrombin and the thrombinantithrombin III complex. Thromb. Res. 36, 397-409, 1984.
12.
JAFFE, E.A., NACHMANN, R.L., BECKER, C.G. and MINICK, C.R. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52, 2745-2756, 1973.
13.
HUNTER, N.R., DAWES, J., MacGREGOR, I.R. and PEPPER, D.S. Quantitation by radioimmunoassay of thrombospondin synthesised and secreted by human endothelial cells. Thromb. Haemostas. 52, 288-291, 1984.
14.
MacGREGOR, I.R. and PROWSE, C.V. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thromb. Res. 2, 461-474, 1983.
15.
DOSNE, A.M., DUPUY, E. and BODEVIN, E. Production of a fibrinolytic inhibitor by cultured endothelial cells derived from human umbilical vein. Thromb. Res. 12, 377-387, 1978.
16.
van MOURIK, J-A., LAWRENCE, D.A. and LOSKUTOFF, D.J. Purification inhibitor of plasminogen activator (antiactivator) synthesised by endothelial cells. J. Biol. Chem. 259, 14914-14921, 1984.
17.
PHILIPS, M., JUUL, A-G. and THORSEN, S. Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex. Biochim. Biophys. Acta, 802, 99-110, 1984.
18.
RIJKEN, D.C., van HINSBERGH, V.W.M. and SENS, E.H.C. Quantitation of tissue-rype plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay. Thromb. Res. 11, 145-153, 1984.
19.
LEVIN, E.G., MARZEC, V., ANDERSSON, J. and HARKER, L.A. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. J. CLIN. INVEST. 74, 1988-1995, 1984.
20.
DEL VECCHIO, P.J. and SMITH, J.R. Aging of endothelium in culture: decrease in angiotensin converting enzyme activity. Cell Biol. Int. Rep. 5, 379-384, 1982.
21.
AGER, A., GORDON, J.L., MONCADA, S., PEARSON, J.D., SALMON, J.A. and TREVETHOCK, M.A. Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial cells and smooth muscle cells. J. Cell. Physiol. 2, 9-16, 1982.
22.
FRY, G., PARSONS, T., HOAK, J., SAGE, H., GUIGRICH, R-D., ERCOLANI, L., NGHIEM, D. and CZERVIONKE, R. Properties of cultured endothelium from adult human vessels. Arteriosclerosis, 4, 4-13, 1984.
of an
BRIEF
COMMUNICATION
THE USE OF HUMAN ENDOTHELIAL CELLS CULTURED IN FLAT WELLS AND ON MICROCARRIER BEADS TO ASSESS TISSUE PLASMINOGEN ACTIVATOR AND FACTOR VIII RELATED ANTIGEN RELEASE
C.V. Prowse', N.R. Hunter', I.R. MacGregor', J.Dawed and D.S. Pepper' 'Scottish National Blood Transfusion Service Headquarters Unit Laboratory and %RC/SNBTS Blood Components Assay Group, 2 Forrest Road, and 'S-E Regional Blood Transfusion Centre. Royal Infirmary, Edinburgh, Scotland. M.M.
McArthur',
(Received 17.7.1985; Accepted in revised form 22.11.1985 by Editor M.J. Seghatchian)
INTRODUCTION Tissue plasminogen activator (t-PA) has been localised in vascular endothelium (l-3) and is released into the circulation following a variety of Since some of these stimuli also stimuli (reviewed by Prowse and Cash, 4). elevate t-PA in models such as perfused pig ear and rat hind limb (5,6) we were interested in investigating if cultured endothelium possesses the capacity to respond to agonists which are active in the whole organism or in isolated organs. Pilot studies on endothelial cells cultured in flat wells (7) indicated that the increased cell surface to medium volume ratio obtained with microcarrier cultures might be more suited to such experiments (8-10) and in this comunication we describe a comparison of the basal secretion of t-PA and factor VIII related antigen (FVIIIR:Ag) in flat well and microcarrier cell cultures and present preliminary data on agonist induced release.
MATERIALS AND METHODS Tissue culture ware was obtained from Costar, via Northumbria Biochemicals, UK. Cell culture media and supplements were from Flow Laboratories, Irvine, UK. Calcium ionophore A23187, bovine serum albumin (BSA) , crystal violet, trypan blue, cycloheximide, actinomycin D and &isopropyl fluorophosphate (DFP) were supplied by Sigma, Poole, UK. Triton X-100, glucose and citric acid were products of BDH, UK. Dimethylsulphoxide (DMSO) was obtained from Aldrich, UK. Cytoaex 3 microcarrier beads were purchased
Part of this work was presented Fibrinolysis, Venice, 1984. KEY WORDS:
at the 7th International
Congress on
Tissue plasminogen activator; factor VIII related antigen; human endothelial cell cultures; microcarrier beads; drugs.
581
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from Pharmacia, Sweden. Fibronectin was obtained from the Protein Fractionation Centre, Edinburgh, UK, and human serum from the Scottish National Blood Transfusion Service. Human c( thrombin was prepared as described previously (11). Endothelial Cell Culture: Endothelial cells were isolated from human umbilical cord veins (HUVEC) by the method of Jaffe et al (12) and cultured and characterised as described previously (13). HUVEC were grown to confluency on Cytodex 3 microcarrier beads by the method of Busch et al (9). Flat Well Cultures: Growth medium was removed from confluent endothelial cells cultured in 9.6 cm2 wells and, following two washes with Medium 199, modified (M 199), was replaced with 1 ml of conditioning medium (CM) consisting of M 199 containing 1 % w/v BSA plus any agonist or control vehicle to The wells were incubated at 37OC for 3,6 or 24 hr then the CM was be tested. removed, centrifuged for 6 min at 8,000 g and the supernatant stored at -40°C prior to radioimmunoassay (RIA) for t-PA and FVIIIR:Ag. The cell monolayer was washed and lysed with 0.5% w/v Triton X-100 in 0.01 M Tris-HCl, pH 8.2 then treated in the same way as the CM sample. Microcarrier Perfusion: 27ml aliquots of washed microcarrier beads covered by endothelial cells ( 1 10 cells/ml of packed beads) were placed in 0.7 cm diameter plastic chromatography columns containing 70 pm pore size filters (Amicon Wright, UK) and were perfused with CM at 0.2 ml/min at 37OC for PO min to obtain basal secretory values. Then agonists were added in a 1 ml pulse or continuously throughout the subsequent perfusion. 0.2 ml fractions of effluent were collected and assayed for t-PA antigen and FVIIIR:Ag. Microcarrier Incubation: Washed microcarrier beads covered by confluent endothelial cells were suspended in an equal volume of CM and 2 ml aliquots transferred to plastic tubes (Sarstedt, UK) where any agonist to be tested was already present in a volume of 50 ~1. The beads were maintained in suspension on a shaking table (Janke and Kunkel, W Germany) at 37OC and 0.5 ml bead-free CM was removed at various times with replenishment by fresh CM. Viability, was assessed by the ability to exclude trypan blue. CM and lysates were prepared and stored as for flat well cultures prior to assay. t-PA RIA: This was performed exactly as described previously, using 50 ~1 test samples in duplicate (14). The working range of the assay was l-80 ng/ml of test sample. FVIIIR:Ag RIA: The RIA was a conventional competition assay using a polyclonal antibody raised in rabbits and an immobilised second antibody separation system. FVIIIR:Ag was iodinated by the Iodo-Gen method to a specific activity of 400 KBq/pg. The assay had a working range of l-100 ng/ml of test sample based on a normal plasma pool content of 10 pg FVIIIR:Ag/ml. Miscellaneous: The cell count in microcarrier cultures was determined by counting nuclei in 0.1 M citric acid containing 0.1% w/v crystal violet and viability was checked by measuring the exclusion of a 2% w/v trypan blue solution (9).
RESULTS t-PA standard curves were constructed using assay buffer or CM (prior to conditioning) or lysing buffer as diluent and the three curves were
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Recovery experiments were performed in two ways. Firstly superimposable. purified t-PA was added at a range of concentrations to cell free CM which had been in contact with confluent HUVEC for 24 hr, and the CMs were assayed for Recoveries averaged 74% and were greater when the CM was t-PA antigen. When t-PA was inactivated by diluted prior to addition of purified t-PA. treatment with DFP before addition to CM the recoveries approached 100%. These results suggested.that CM contained components which modified t-PA to a form less readily recognised in the RIA and that the modification required a free active site. Secondly, when t-PA or DFP inactivated t-PA was added to confluent HUVEC and the resultant cell free CM was harvested and assayed after 24 hr an average recovery of 20% and 40% respectively was obtained. The low recovery of purified t-PA added to HWEC cultures appears partly due to adsorption on cell surfaces and partly to its modification to a form less well detected by the RIA. Whether t-PA secreted by HUVEC behaves in a similar fashion is presently being investigated. t-PA antigen accumulated in the CM of HWEC cultures in Flat Well Cultures: a time dependent fashion and was also detectable in the cell lysates. The expressed antigen was shown to be a synthetic product of the cells ince pre-5 treatment with cycloheximide at 2 pg/ml or actinomycin D at 4 x 10 M resulted in a marked reduction in both secreted and intracellular
120. t-PA antigen secreted
60
n 0
12 TIME (HOURS) FIG. 1
Relationship between passage number of HWEC line and secreted levels of t-PA
5 cell
??- ??= ~3; A- A= P6; ??- m= P8; 0 - o= P12; 0-o = P18 where P = passage number and each passage represents two population doublings
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concentrations of t-PA. In early passage cells (P3 and 4) lo-20 ng t-PA/lo' cells was typically secreted over a 24 hr period. The increase of secreted t-PA antigen as a function of time is shown in Fig.1. The rate of basal secretion was greater in late passage cultures with all cell lines examined. In all subsequent experiments control and treated cells were derived from the same cell line at the same passage number. The effect of thrombin at 1 u/ml At 3 hr after upon t-PA secretion was tested in five separate experiments. addition of thrombin secreted t-PA was 92 k 26% of the control value (3i2 S.D.) while at 24 hr it was 126 2 23% of the control valce.
treated 10 u/ml Calcium tration
t-PA secretion at 3,6 and 24 hr was similar in control and thrombincells while short-term secretion of FVIIIR:Ag was stimulated by51 and thrombin and also at the early times by calcium ionophore (10 M) . ionophore reduced both t-PA secretion and the intracellular concenof t-PA over the 24 hr period by approximately 80%.
Microcarrier Perfusion: Microcarrier beads covered by confluent HUVEC were t-PA release over the first 60 min perfused over 5 hr with CM at 0.2 ml/min. was erratic, but after this time a steadier rate of secretion was observed. The rate of this steadier basal secretion of t-PA antigen in 15 perfusion experimgnts with 3 cell lines all at passages between 5 and 9 was 30 ?r 27 pg t-PA/10 cells/min (mean k SD). This was quantitatively similar6to the typical rate of secretion in flat well cultures of 42 pg t-PA/10 cells/min, but because of the high cell surface to medium volume ratio obtainable in microcarrier perfusion, this system can be used to detect short-term changes in t-PA concentration.
-30
0.1 t-PA antigen secretec I
FVlll R : Ag * m‘? secretea LU (ng /lObcells
c
( ng /106cells)
.lO
0
I-
0
,O
8
20
40
60
80
TIME lMlN 1
FIG. 2 Secretion of t-PA and FVIIIR:Ag by perfused HUVEC attached _5 to microcarrier beads. M. Effects of calcium ionophore A23178 at 10 Time 0 follows a 60 min equilibrating perfusion period. t-PA,
O-0;
FVIIIR:Ag,
.---..
Hatched bars indicate presence of ionophore
1
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ENDOTHELIAL CELL t-PA
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Basal secretion of FVIIIR:Ag was also detectable in the microcarrier In six expegiments using HWEC at passage 5 the basal perfusion system. secretion was 13.0 f 12.1 ng/lO cells/min (mean f SD) and E'VIIIR:Ag would be undetectable over such short time courses in flat well cultures. Figure 2 shows basal secretion of t-PA and E'VIIIR:Ag by perfused cells on microcarriers and the transient stimulation of F'VIIIR:Ag release by calcium ionophore which was seen in each of three separate experiments. Because of the variation in basal secretion during Microcarrier incubation: perfusion experiments a time course of basal synthesis and secretion of t-PA and FVIIIR:Ag was set up over O-180 min using a discontinuous batch perfusion system and both antigens were secreted in approximately a linear fashion with respect to time although the earliest (5 min) sample invariably contained more antigen than expected from the secretion versus time curve. The amount of t-PA and6E'VIIIR:Ag typically secgeted by an early passage cell line was 15 pg/lO cells/min and 14 ng/lO cells/min respectively. Calcium ionophore stimulated F'VIIIR:Ag release at 10 min (157% of control 10 min value) while t-PA secretion was inhibited.
DISCUSSION By using an RIA we have been able to quantify t-PA antigen produced by cultured human endothelium in conditions where no biological activity can be detected because of the presence of excess plasminogen activator inhibitor (15-17). The t-PA was shown to be a synthetic product by using protein synthesis inhibitors. Recovery experiments indicated that t-PA with a free active site forms inhibitor complexes less readily detectable in the RIA as reported by Rijken et al (18) although the immunoassay of Levin et al detected both free and inhibited t-PA equally well (19). Recovery of added t-PA from cell cultures suggested that t-PA may be adsorbed to cell surfaces, and if this also occurred with native t-PA an underestimate of secretory rates would result. Further experiments are needed to establish the disposition of t-PA after secretion by cultured HUVEC. We observed differences in basal secretion of t-PA between cell lines and an increase with gigher passage number. In early passage cells our values of lo-20 ng/lO cells/24 hr corn qred with published values for primary or first passage HUVEC of about 5 ng/lO /24 hr (18,19). Alterations in expression of endothelial metabolites following repeated subcultivation has been reported for angiotensin converting enzyme and prostacyclin (which decrease) while 5'-nucleotidase increases (20-22). Conversely the secretion of some metabolites such as thrombospondin are unaffected by serial subcultivation (13). The reason for induction of t-PA synthesis and secretion in late passage cells is unknown but merits further investigation as a model of occurrences in vivo in areas of high endothelial turnover during chronic endothelial injury. Nor is it known whether concomitant changes in the synthesis of t-PA inhibitors occur. We have shown here that HUVEC cultured on microcarrier beads secrete t-PA and F'VIIIR:Ag at confluence at similar rates to HUVEC grown on conventional tissue culture plastic surfaces, but because of the former's much greater cell surface to medium volume ratio we could detect the release of these proteins over a considerably shorter interval showing that the microcarrier cultures are theoretically suitable for measuring short term release reactions. While calcium ionophore was shown to promote rapid release of
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FVIIIR:Ag in the perfused microcarrier cell system we have been unable to demonstrate release of t-PA by a range of agonists (including DDAVP, platelet activating factor and bradykinin) which have been shown by others to promote its release in intact animals and isolated organs (unpublished observations). Other agents which increase t-PA secretion may do so by effects upon Thus thrombin in the presence of serum induced t-PA protein synthesis. release from HUVEC with a 6 hour lag and a maximum effect at 16 hours (19) and Therefore, the such studies are more suited to flat well culture systems. use of flat well and microcarrier ccl 1 cultures should be seen as complementary for investigating modulators of t-PA secretion.
ACKNOWLEDGEMENTS Purified We thank Miss S. Maguire for expert technical assistance. t-PA was kindly provided by Dr. D. Collen, Leuven, Belgium, and Dr. M. We acknowledge the support of the Medical Einarsson, Kabi Vitrum, Sweden. Research Council (Project Grant G8217210SB) to C.V.P. and I.R. MacG., (Programme Grant ~G8218470) to J.D. and D.S.P. and the British Heart Foundation (BHF Grant 82/13) to I.R.MacG. and D.S.P.
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