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TISSUE-PLASMINOGEN ACTIVATOR STIMULATES ENDOTHELIAL CELL MIGRATION IN WOUND ASSAYS A.L.INYANG*
and G.TOBELEM
INSERM U150, Hopital Lariboisiere, 75475 Paris, France Received
August
16,
1990
SUMMARY: The ability of tissueplasminogenactivator (tPA) to induce human umbilical vein endothelial (HUVE) cell migration was studiedusing an in vitro, serum-freewound assay system. At pharmacological doses,tPA stimulated HUVEcell migration dosedependently-Treatmentof cells with epsilon amino caproic acid (EACA) to detach cellsurface and extracellular matrix bound plasminogenwhich could lead to plasmin generation,resultedin increasedHUVEcell migrationon stimulationwith tPA.Plasminogen activator inhibitor- 1 (PAI- l), a natural plasminogenactivator inhibitor, abolished tPAinducedHUVEcell migration.Theseresultsdemonstratefor the first time that tPA is capable of stimulating endothelial cell migration in wound assaysand this effect is susceptibleto PAI- inhibition, O1990 Academic Press, ~nc.
INTRODUCTION. In large blood vessels, the endothelial cell responseto lesions involves migration to cover up denudedareasand proliferation.This responseis regulated by an interplay of a multitude of factors, the most important ones being the angiogenic growth factors such as basic fibroblast growth factor (bFGF) and the endogenous proteases such as the plasminogen activators (PAS) (l-3). This phenomenon can be reproduced in vitro by mechanically wounding a confluent monolayer of cultured endothelial cells (45). A host of studieshave demonstratedthe involvement of PAS in endothelialcell migration during wound healing,angiogenesisand other invasive processes (6-8). Of the two types of PAS found in endothelial cells, urokinase-type PA (uPA) is primarily linked with invasive processeswhile tissue-plasminogenactivator (tPA) is basically associatedwith the fibrinolytic system(9). These plasminogenactivators are inhibited by plasminogen activator inhibitor-type 1 (PAI-1; ref.10). The production, secretionand binding to cell surfacereceptorsof theseproteasesand their inhibitors in large vessels and capillary endothelial cells have been extensively studied (11-13). In vitro,primary and early cultures of human umbilical vein endothelial cells (HUVEcells) producepredominantly tPA (14) althoughthe production of uPA hasalsobeenreportedin late cultures (15) .ThesePAS activate plasminogen(PLG) to form plasmin, a non specific serineprotease,that contributesto cell migration through basementmembranedegradation
*Correspondenceand reprint requestshould be addressedto Dr.A.L.Inyang,The Whittier Institute for DiabetesandEndocrinology,9894GeneseeAvenue,La Jolla,CA 92037. OLM6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and the activation of other proteasesinvolved in this process (1,3,16,). However, the role of tPA,which is the only form of PAS found so far in endothelial cells of large vesselsof humans (17), in the migratory processesof the endothelial cells remainsunclear. Most studiesindicate that only uPA is involved in EC migration in wound situations (4,18), thus begging the questionsof how large vessellesionsare resealedin vivo and what the role of tPA in EC migration is in suchsituations. In this paper, using the wounded monolayer model, we demonstratethat tPA,in pharmacologicaldoses,caninduce HUVEcell migration in a systemthat hasbeendepleted of PLG, and that PA1 inhibits this effect . MATERIALS AND METHODS. w ts and Mate rialsr Tissueculture reagentsfrom Boerhinger Mannheim ( France > were use\.Twelve-multiwell plateswere obtained from Costar (Cambridge&IA ). Phorbol I%-Myristate 13-acetate(PMA), aprotininbovine serum albumin (BSA), and gelatin were purchasedfrom Sigma (St. Louis,MO ).Basic fibroblast growth factor (bFGF) was made by British Biotecnology(England).Recombinant tissueplasminogen activator (rt-PA)was a kind donation from Boerhinger Ingelheim (France).Dr. D.Collen of the Centre for Thrombosis and Vascular Research,Leuven,Belgiumkindly donatedthe recombinantplasminogenactivator inhibitor1 (rPAI-1 )which wasreactivated as previously described(l9).The specific activity of the rPAI-1 was about 15 x 104U/ml.fibronectin was a product of IBF (France) while epsilon amino caproic acid (EACA) waspurchasedfrom Aldrich. QQ: Human umblical vein endothelial cells were preparedaccording to the method of Jaffe (2O).The cells were cultured at 37 oC in a humidified atmospherewith 5% CO2 in Medium 199containing 5OU/ml penicillin and 5Oug/mlfungizone supplementedwith 20% fetal calf serum.Cells were grown on gelatin -coated plates and used after the first passage.Theendothelialnatureof the cellswas ascertainedby their cobblestoneappearance in confluent cultures and the presence of factor VIII&on Willebrand factor antigen demonstratedusingindirect immunofluorescence Wound: HUVE cell migration was monitored using the wound assay initially described by Burk (21).Briefly,about 18x1@ cells were seededon fibronectin-coated polycarbonate lamelle ( 10.5 x22 mm,NUNC Inc. Nashville) and allowed to grow for 48 hours in a 35mm culture dish usingM199+20% fetal calf serum.Thiscell density allowed the cells to be closely packed as found in blood vessels.Cellswere washedtwice with Ml99 before and after wounding.The wounded cells were then incubated in the assay medium (M199+0.4%BSA+0.2% gelatin) with the specifiedtest substances,and left for 30 hours.At the end of incubation ,the cells were washedonce with cold PBS,fixed with 95% methanol and stainedwith Giemsa.Cellswere counted at 100x magnification using a light microscope.Resultswere expressedas the mean number of migrating cells per field f. standarddeviationA minimumof 4 fields were countedper assayand eachexperiment was repeated3 to 6 times. Cvtotoxicitv assay;The cytotoxic effect of tPA and PA1 on HUVEcells in the conditions describedin this study,wasmeasuredby the trypan blue exclusion method.Cellviability at the concentrationsandconditions describedin this study wasnever below 93%. RESULTS.Effeet
ion: The effect of tPA on endothelial cell
migration was assessed in a serum-freesystem.To study the direct role of this proteaseon endothelial cell migration in a plasminogen-freesystem.At pharmacologicconcentrations of 0.2 to 2.0 ug/ml, tPA induced HUVEcell migration in a dose-dependentmanner (Fig. 1). The extent of migration induced with 2ug/ml tPA wascomparableto that with 25ngfml bFGF but lessthan that with 50 @ml PMA (Fig.2b).Cells stimulated with tPA did not migrate as far as those treated with PMA, but migration was spreadmore all along the wound front. (our observations). An additive effect resulted from the addition of tPA to 1327
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jig/ml tPA &J. Dose-dependent induction of HUVEcell migration by tPA.Confluent monolayers of HUVEcells were wounded with a razor blade,incubated with indicated additions for 30 h,fixed and stained.Migration was quantitated as described in Materials and Methods.Results represent mean + standard deviation.
bFGF,but tPA did not influence appreciably PMA- induced migrations (unpublished results). . .. Inblblhon of tPA induced mkration bv PAI-1: PAI- is a natural inhibitor of tPA produced by endothelial cells and is capable of inhibiting both tPA and UPA (lo), thus regulating 120
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l&&t. Inhibition of tPA-induced HUVEcell migration by PAI-l.Confluent monolayers of HUVEcells were wounded,incubated simultaneously with specified additions and quantitated as described in the legend to Fig.l.(A) control ; (B) tPA (lug/ml) ; (C) tPA (lug/ml) + PAI-1 (0.5 @ml ) ; (D) tPA (lug/ml)+PAI-1 (lug/ml) ; (E) PAI- (lug/ml). FiP-2b, Inhibition of PMA and bFGF induced HUVEcell migration by PALl.Cells were treated as described in the legend to Fig.l.Additions were made simultaneously as shown.
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&j, Inhibition of tPA induced HUVEcell migration by EACA .Cells were wounded and migration quantified as described in Materials and Methods.Additions were made simultaneously EACA wasincubatedwiith the cells throughoutthe duration of the assay..(A)control ; (B) tPA (2ug/ml) ; (C) tPA (2uglml)+EACA (500 @ml) ; && Effect of EACA treatment on basal and tPA induced HUVEcell migration. Confluent monolayers of HUVEcells were wounded and migration assess& as described in Materials and Methods.Some cells were incubated at 4 oC with 20 mM EACA for 20 min,washed twice with cold PBS before being wounded and quantified for migration in the
presence of tPA. their activity. At a concentration of OS-lug/ml, PA1 inhibited both the basal and tPA induced migrations (Fig.2a). Since bFGF and phorbol estersare very strong inducersof endothelial cell migration and PA synthesis (6,18), we examined the effect of PA1 on migration induced with theseagentsour results show that PAI antagonizedthe migratory effects of bFGF and PMA on HUVEcells (Fig2b). This inhibition was more pronounced with bFGF-induced migration than that inducedby PMA. Eff fA 1 ‘on : We examinedthe effect of EACA,a known protease-inhibitor,ontPA-induced migration.At a concentrationof 5OOug/ml,EACA almost completely blocked HUVEcell migration induced with tPA. (Fig.3). We also studiedthe role of PLG in our assaysystemIt could be arguedthat in our assaysystemour cells still had somePLG boundto either the cell surfaceor the extracellular matrix (ECM) (22) and that our resultscould thus be interpreted to be an effect of plasmin (3) and not a direct tPA effect. To resolve this,the cells were washed with EACA for 20 minutes to remove surface- and ECM-bound PLG (22) before wounding,and migration was then assayedwith tPA. Figure 4 showsthat EACA treatment did not reduce the number of migrating cells aswould have beenthe caseif the actionsof tPA dependedentirely on the involvement of PLG.Contrary to our expectations,therewas an increasein the number of migratingcells (after EACA treatment) in basalandtPA stimulatedcells. DISCUSSION. Several studies have demonstrated the participation of uPA in EC migration in a wound assay, in angiogenesisand in other invasive processes(18). 1329
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Endothelial cells are known to contain both uPA and tPA in various proportions (9). However,using immunochemicaltechniques,inthe endotheliumof humanlarge vessels,it hasbeendemonstratedthat only tPA could be detected(17), although the presenceof uPA hasbeendemonstratedin a variety of non vascular tissuesusingthis technique(2). Hence, in such vessels, where the tissue form of the activator is predominant, tPA would be expectedto contribute to the migratory activities of thesecells in wound conditions. In this study,we have usedan in vitro model to demonstratefor the first time that tPA can stimulate HUVEcells to migrate in wound assays.Thiseffect is a dose-related effect which is also susceptible to the inhibitory effect of PAI. Many studies have demonstratedthat the induction of migration by PAS in invasive processesis dependenton the presenceof PLG and the subsequentformation of plasmin (1,3,16). By using a system which was serum-free, we reduced immenselythe contribution of plasmaPLG and other serumfactors which would otherwise have madeinterpretation of our data complicated. However, depleting the serumof PLG- or using a serum-freemediumdoesnot necessarily mean a PLG-free system since PLG binds to both EC and ECM (22). EACA treatment (22) stripsthe cells of most of the boundPLG thus providing a systemfor examining the direct effect of tPA on EC migrationAs cell migration could still be stimulatedafter this treatment,weconcludedthat tPA’s effect on HUVEcell migration could be by a mechanism which wasoperationaleven in the presenceof little or no plasmingeneration. Presently, the actual contribution of tPA in cell migration remains underscored.Moststudiesindicate that tPA doesnot participate in cell migratory functions (2,4,16) In the system studied here, exogenously addedtPA could induce migration of woundedcells.In vivo, suchstimulation of cell migration by tPA could be complimentary to that mediated by uPA.The exact mechanismof the observed effects of tPA on EC migration in wound situationsis not known but may be related to receptor activation and cytoskeletal reorganisationafter wounding (4,7). The involvement of receptor activation in this systemis presently being studied.It shouldbe bornein mind that the situationfound in vivo is a highly complicated one involving the interaction of a wide range of enzymesgrowth factors,chemoattractantsandproteaseinhibitors all regulating eachothers activity.In this veinalthough we could reduce to the the barest minimum the likely contribution of plasmin by lowering PLG concentration,we do not know how tPA,at the concentration usedhere, would affect other proteasessuch as the metalloproteinasesand the glycasesthat participate in cell migration. However,as complex as the situation may be,the potential of tPA as an inducer of EC migration will dependon the cells ability to produce and interact with this substancethrough its cell surface receptors, as has been described for uPA mediated cell migration (7). This criteria has been satisfied by tPA (14,28). PAI- is a natural inhibitor of tPA. It is presentin the ECM of both bovine aortic ECS and HUVEcells (10). We have usedPAI- to inhibit the actionsof tPA in our system PMA and bFGF induced migration of HUVEcells were also susceptibleto PAI mediated inhibition.These substancesstimulate PA production in endothelial cells (6,2.5,26). Although PAI- also inhibits uPA, in our systemvery little uPA is presentasonly tPA is 1330
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produced in early cultures of HWEcells (14) ; thus we suggest that the observed actions of PA1 on bFGF-and PMA-induced migrations may be due,in part to tPA inhibitionHowever, we are cautious about this interpretation as it is not known if wounding could induce these cells to produce uPA,which would then stimulate the cells to migrate.This possibility is currently being examined.That basal migration in wounded cells was also inhibited by PA1 could be due to the inhibition of PAS released by the cells after wounding or formed from growth factors which is released on wounding ($27). EACA,a non-specific protease inhibitorantagonized tPA induced HWEcell migration in our assay. Some studies indicate that EACA acts by plasmin inhibition (8). Since we used a system that could not generate plasmin to any appreciable extent, we suggest that this inhibition is a result of EACA’s effect on the lysine-binding sites of tPA, as shown previously (28). In conclusion,we have demonstrated that tPA is capable of stimulating HUVEcell migration in wound assays. We suggest that tPA may play some role in EC migration, at least in large vessels,where it is produced,is readily secreted on stimulation and can bind to its cell surface receptor; requirements necessary for induction of migration (7). Such a role could be complimentary to that of uPA in the healing of large vessel lesions and deserves further studies.
ACKNOWLEDGMENT:
This study was supported by the Fondation pour la Recherche
Medicale for ALI.
REFERENCES l.Moscatelli, D.& Rifkin, D.B., (1988) Biochim et Biophy Acta. 948,67- 85. 2.Dano, K., Andreasen, P.A., GrondahlHansen, J., Kristensen, P., Nieelsen, L.S., &&river, L.( 1985) Adv.Cancer Res.44,139-226. XMignatti, P., Tsuboi, R., Robbins, E. & Rifkin, D.B.( 1989). J.Cell.Biol. 108, 67 l682. 4.Pepper, M.S., Vassalli, J.-D, Montesano, R.& Grci, L.(1987). J.Cell Biol. 105, 2535 2541. S.McNeil, P.L., Mathurkrishan, L.,Warder, E.& D’Amore, P.A.(1989). J.Cell.Biol. 109,81 l- 822. &Sato, Y. & Rifkin, D.B. (1988).J.Cell.Biol. 107, 1199-1205. 7.Ossowski, L.(1988). J.Cell.Biol. 107, 2437-2445. g.Inger, D.E. & Folkman, J.(1989) J.Cell.Biol. 109, 317-330. 9.Collen D., Lijnen, H.R., Todd, P.A. & Goa, K.L. (1989) Drug 38, 346-388. lO.Sprengers, E.D. & Kluft, C.(1987) Blood 69, 381-387. ll.Aerts, R.J., Gillis, K. & Pannekoek, H.(1989). Thromb.Haemostas.62, 699-703. 12.Hajjar, K.A., Hamel, N.M., Harpel, P.C.& Nachman, R.L.(1987). J.Clin.Invest. 80, 1712-1719. 13.Sakata, Y., Okada, M., Noro, A.& Matsuda, M(1988). J.Biol.Chem. 263, 19601969. 14.van Hinsberg, V.W.M., Binnema, D., Scheffer,M.A., Spergers,E.D., Kooistra,T. & Rijken, D.C.(1987). Artherosclerosis 7,389-399. lS.Booyse, F.M., Schneinbuks,J., Lin, P.H., Traylor, M.& Bruce, R.(1988).J. Biol.Chem 263, 15129-15138. ldOssowski, L., Quingley, J.P.& Reich, E.(1975). In Protease and Biological Controls. Cold Spring Harbour. pp 109. 17.Rijken, D.C., Wijngaards, G.&z Welbergen, J.(1980). Thromb.Res.18, 815-830. 1331
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l&Montesano, R., Vassalli, J.D., Baird, A., Gullemin, R.& Grci,L.(1986) ProcNatl. Acad.Sci.USA 83,7297-7301. 19.Alessi,M.C.,DeClerck, J.P., DeMol, M., Nelles, L.& Collen,D.(1988). Eur.J. B&hem, 175.531-540. tO.Burk, R.R.(1973). Proc.Natl.Acad.Sci.USA. 70, 369-372. 21.Jaffe, E.A., Nachman, R.L., Becker, C.G.& Minick, C.R.(1973) J.Clin.Invest. 52, 2745-2756. 22.Hajjar, K.A., Harpel,P.A.,Jaffe, E.A.&.Nachman, R.L.(1986).J.Biol.Chem.261, 11656-l 1662. 23.Schleef, R.R.& Birdwell, CR. (1982). Exp.Cell.Res. 141, 503-508. 24.Levin, E.G.& !&tell, L.( 1988). J.Biol.Chem. 263, 9360-9365. 2S.Presta, M., Maier, J.A.M.& Ragnotti, G.(1989). J.Cell.Biol. 109, 1877-1884. 26.Saksela, O., Moscatelli, D.& Rifkin, D.B.(1987) J.Cell.Biol. 105,957-963. 27Plaumenhaft, R., Moscatelli, D.,Saksela, O.& Rifkin, D.B.(1989). J.Cell.Physiol. 140, 75-81. 2&Beebe,D. (1987). Thromb.Res. 46, 241-254.
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TISSUE-PLASMINOGEN ACTIVATOR STIMULATES ENDOTHELIAL CELL MIGRATION IN WOUND ASSAYS A.L.INYANG*
and G.TOBELEM
INSERM U150, Hopital Lariboisiere, 75475 Paris, France Received
August
16,
1990
SUMMARY: The ability of tissueplasminogenactivator (tPA) to induce human umbilical vein endothelial (HUVE) cell migration was studiedusing an in vitro, serum-freewound assay system. At pharmacological doses,tPA stimulated HUVEcell migration dosedependently-Treatmentof cells with epsilon amino caproic acid (EACA) to detach cellsurface and extracellular matrix bound plasminogenwhich could lead to plasmin generation,resultedin increasedHUVEcell migrationon stimulationwith tPA.Plasminogen activator inhibitor- 1 (PAI- l), a natural plasminogenactivator inhibitor, abolished tPAinducedHUVEcell migration.Theseresultsdemonstratefor the first time that tPA is capable of stimulating endothelial cell migration in wound assaysand this effect is susceptibleto PAI- inhibition, O1990 Academic Press, ~nc.
INTRODUCTION. In large blood vessels, the endothelial cell responseto lesions involves migration to cover up denudedareasand proliferation.This responseis regulated by an interplay of a multitude of factors, the most important ones being the angiogenic growth factors such as basic fibroblast growth factor (bFGF) and the endogenous proteases such as the plasminogen activators (PAS) (l-3). This phenomenon can be reproduced in vitro by mechanically wounding a confluent monolayer of cultured endothelial cells (45). A host of studieshave demonstratedthe involvement of PAS in endothelialcell migration during wound healing,angiogenesisand other invasive processes (6-8). Of the two types of PAS found in endothelial cells, urokinase-type PA (uPA) is primarily linked with invasive processeswhile tissue-plasminogenactivator (tPA) is basically associatedwith the fibrinolytic system(9). These plasminogenactivators are inhibited by plasminogen activator inhibitor-type 1 (PAI-1; ref.10). The production, secretionand binding to cell surfacereceptorsof theseproteasesand their inhibitors in large vessels and capillary endothelial cells have been extensively studied (11-13). In vitro,primary and early cultures of human umbilical vein endothelial cells (HUVEcells) producepredominantly tPA (14) althoughthe production of uPA hasalsobeenreportedin late cultures (15) .ThesePAS activate plasminogen(PLG) to form plasmin, a non specific serineprotease,that contributesto cell migration through basementmembranedegradation
*Correspondenceand reprint requestshould be addressedto Dr.A.L.Inyang,The Whittier Institute for DiabetesandEndocrinology,9894GeneseeAvenue,La Jolla,CA 92037. OLM6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and the activation of other proteasesinvolved in this process (1,3,16,). However, the role of tPA,which is the only form of PAS found so far in endothelial cells of large vesselsof humans (17), in the migratory processesof the endothelial cells remainsunclear. Most studiesindicate that only uPA is involved in EC migration in wound situations (4,18), thus begging the questionsof how large vessellesionsare resealedin vivo and what the role of tPA in EC migration is in suchsituations. In this paper, using the wounded monolayer model, we demonstratethat tPA,in pharmacologicaldoses,caninduce HUVEcell migration in a systemthat hasbeendepleted of PLG, and that PA1 inhibits this effect . MATERIALS AND METHODS. w ts and Mate rialsr Tissueculture reagentsfrom Boerhinger Mannheim ( France > were use\.Twelve-multiwell plateswere obtained from Costar (Cambridge&IA ). Phorbol I%-Myristate 13-acetate(PMA), aprotininbovine serum albumin (BSA), and gelatin were purchasedfrom Sigma (St. Louis,MO ).Basic fibroblast growth factor (bFGF) was made by British Biotecnology(England).Recombinant tissueplasminogen activator (rt-PA)was a kind donation from Boerhinger Ingelheim (France).Dr. D.Collen of the Centre for Thrombosis and Vascular Research,Leuven,Belgiumkindly donatedthe recombinantplasminogenactivator inhibitor1 (rPAI-1 )which wasreactivated as previously described(l9).The specific activity of the rPAI-1 was about 15 x 104U/ml.fibronectin was a product of IBF (France) while epsilon amino caproic acid (EACA) waspurchasedfrom Aldrich. QQ: Human umblical vein endothelial cells were preparedaccording to the method of Jaffe (2O).The cells were cultured at 37 oC in a humidified atmospherewith 5% CO2 in Medium 199containing 5OU/ml penicillin and 5Oug/mlfungizone supplementedwith 20% fetal calf serum.Cells were grown on gelatin -coated plates and used after the first passage.Theendothelialnatureof the cellswas ascertainedby their cobblestoneappearance in confluent cultures and the presence of factor VIII&on Willebrand factor antigen demonstratedusingindirect immunofluorescence Wound: HUVE cell migration was monitored using the wound assay initially described by Burk (21).Briefly,about 18x1@ cells were seededon fibronectin-coated polycarbonate lamelle ( 10.5 x22 mm,NUNC Inc. Nashville) and allowed to grow for 48 hours in a 35mm culture dish usingM199+20% fetal calf serum.Thiscell density allowed the cells to be closely packed as found in blood vessels.Cellswere washedtwice with Ml99 before and after wounding.The wounded cells were then incubated in the assay medium (M199+0.4%BSA+0.2% gelatin) with the specifiedtest substances,and left for 30 hours.At the end of incubation ,the cells were washedonce with cold PBS,fixed with 95% methanol and stainedwith Giemsa.Cellswere counted at 100x magnification using a light microscope.Resultswere expressedas the mean number of migrating cells per field f. standarddeviationA minimumof 4 fields were countedper assayand eachexperiment was repeated3 to 6 times. Cvtotoxicitv assay;The cytotoxic effect of tPA and PA1 on HUVEcells in the conditions describedin this study,wasmeasuredby the trypan blue exclusion method.Cellviability at the concentrationsandconditions describedin this study wasnever below 93%. RESULTS.Effeet
ion: The effect of tPA on endothelial cell
migration was assessed in a serum-freesystem.To study the direct role of this proteaseon endothelial cell migration in a plasminogen-freesystem.At pharmacologicconcentrations of 0.2 to 2.0 ug/ml, tPA induced HUVEcell migration in a dose-dependentmanner (Fig. 1). The extent of migration induced with 2ug/ml tPA wascomparableto that with 25ngfml bFGF but lessthan that with 50 @ml PMA (Fig.2b).Cells stimulated with tPA did not migrate as far as those treated with PMA, but migration was spreadmore all along the wound front. (our observations). An additive effect resulted from the addition of tPA to 1327
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jig/ml tPA &J. Dose-dependent induction of HUVEcell migration by tPA.Confluent monolayers of HUVEcells were wounded with a razor blade,incubated with indicated additions for 30 h,fixed and stained.Migration was quantitated as described in Materials and Methods.Results represent mean + standard deviation.
bFGF,but tPA did not influence appreciably PMA- induced migrations (unpublished results). . .. Inblblhon of tPA induced mkration bv PAI-1: PAI- is a natural inhibitor of tPA produced by endothelial cells and is capable of inhibiting both tPA and UPA (lo), thus regulating 120
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l&&t. Inhibition of tPA-induced HUVEcell migration by PAI-l.Confluent monolayers of HUVEcells were wounded,incubated simultaneously with specified additions and quantitated as described in the legend to Fig.l.(A) control ; (B) tPA (lug/ml) ; (C) tPA (lug/ml) + PAI-1 (0.5 @ml ) ; (D) tPA (lug/ml)+PAI-1 (lug/ml) ; (E) PAI- (lug/ml). FiP-2b, Inhibition of PMA and bFGF induced HUVEcell migration by PALl.Cells were treated as described in the legend to Fig.l.Additions were made simultaneously as shown.
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&j, Inhibition of tPA induced HUVEcell migration by EACA .Cells were wounded and migration quantified as described in Materials and Methods.Additions were made simultaneously EACA wasincubatedwiith the cells throughoutthe duration of the assay..(A)control ; (B) tPA (2ug/ml) ; (C) tPA (2uglml)+EACA (500 @ml) ; && Effect of EACA treatment on basal and tPA induced HUVEcell migration. Confluent monolayers of HUVEcells were wounded and migration assess& as described in Materials and Methods.Some cells were incubated at 4 oC with 20 mM EACA for 20 min,washed twice with cold PBS before being wounded and quantified for migration in the
presence of tPA. their activity. At a concentration of OS-lug/ml, PA1 inhibited both the basal and tPA induced migrations (Fig.2a). Since bFGF and phorbol estersare very strong inducersof endothelial cell migration and PA synthesis (6,18), we examined the effect of PA1 on migration induced with theseagentsour results show that PAI antagonizedthe migratory effects of bFGF and PMA on HUVEcells (Fig2b). This inhibition was more pronounced with bFGF-induced migration than that inducedby PMA. Eff fA 1 ‘on : We examinedthe effect of EACA,a known protease-inhibitor,ontPA-induced migration.At a concentrationof 5OOug/ml,EACA almost completely blocked HUVEcell migration induced with tPA. (Fig.3). We also studiedthe role of PLG in our assaysystemIt could be arguedthat in our assaysystemour cells still had somePLG boundto either the cell surfaceor the extracellular matrix (ECM) (22) and that our resultscould thus be interpreted to be an effect of plasmin (3) and not a direct tPA effect. To resolve this,the cells were washed with EACA for 20 minutes to remove surface- and ECM-bound PLG (22) before wounding,and migration was then assayedwith tPA. Figure 4 showsthat EACA treatment did not reduce the number of migrating cells aswould have beenthe caseif the actionsof tPA dependedentirely on the involvement of PLG.Contrary to our expectations,therewas an increasein the number of migratingcells (after EACA treatment) in basalandtPA stimulatedcells. DISCUSSION. Several studies have demonstrated the participation of uPA in EC migration in a wound assay, in angiogenesisand in other invasive processes(18). 1329
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Endothelial cells are known to contain both uPA and tPA in various proportions (9). However,using immunochemicaltechniques,inthe endotheliumof humanlarge vessels,it hasbeendemonstratedthat only tPA could be detected(17), although the presenceof uPA hasbeendemonstratedin a variety of non vascular tissuesusingthis technique(2). Hence, in such vessels, where the tissue form of the activator is predominant, tPA would be expectedto contribute to the migratory activities of thesecells in wound conditions. In this study,we have usedan in vitro model to demonstratefor the first time that tPA can stimulate HUVEcells to migrate in wound assays.Thiseffect is a dose-related effect which is also susceptible to the inhibitory effect of PAI. Many studies have demonstratedthat the induction of migration by PAS in invasive processesis dependenton the presenceof PLG and the subsequentformation of plasmin (1,3,16). By using a system which was serum-free, we reduced immenselythe contribution of plasmaPLG and other serumfactors which would otherwise have madeinterpretation of our data complicated. However, depleting the serumof PLG- or using a serum-freemediumdoesnot necessarily mean a PLG-free system since PLG binds to both EC and ECM (22). EACA treatment (22) stripsthe cells of most of the boundPLG thus providing a systemfor examining the direct effect of tPA on EC migrationAs cell migration could still be stimulatedafter this treatment,weconcludedthat tPA’s effect on HUVEcell migration could be by a mechanism which wasoperationaleven in the presenceof little or no plasmingeneration. Presently, the actual contribution of tPA in cell migration remains underscored.Moststudiesindicate that tPA doesnot participate in cell migratory functions (2,4,16) In the system studied here, exogenously addedtPA could induce migration of woundedcells.In vivo, suchstimulation of cell migration by tPA could be complimentary to that mediated by uPA.The exact mechanismof the observed effects of tPA on EC migration in wound situationsis not known but may be related to receptor activation and cytoskeletal reorganisationafter wounding (4,7). The involvement of receptor activation in this systemis presently being studied.It shouldbe bornein mind that the situationfound in vivo is a highly complicated one involving the interaction of a wide range of enzymesgrowth factors,chemoattractantsandproteaseinhibitors all regulating eachothers activity.In this veinalthough we could reduce to the the barest minimum the likely contribution of plasmin by lowering PLG concentration,we do not know how tPA,at the concentration usedhere, would affect other proteasessuch as the metalloproteinasesand the glycasesthat participate in cell migration. However,as complex as the situation may be,the potential of tPA as an inducer of EC migration will dependon the cells ability to produce and interact with this substancethrough its cell surface receptors, as has been described for uPA mediated cell migration (7). This criteria has been satisfied by tPA (14,28). PAI- is a natural inhibitor of tPA. It is presentin the ECM of both bovine aortic ECS and HUVEcells (10). We have usedPAI- to inhibit the actionsof tPA in our system PMA and bFGF induced migration of HUVEcells were also susceptibleto PAI mediated inhibition.These substancesstimulate PA production in endothelial cells (6,2.5,26). Although PAI- also inhibits uPA, in our systemvery little uPA is presentasonly tPA is 1330
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produced in early cultures of HWEcells (14) ; thus we suggest that the observed actions of PA1 on bFGF-and PMA-induced migrations may be due,in part to tPA inhibitionHowever, we are cautious about this interpretation as it is not known if wounding could induce these cells to produce uPA,which would then stimulate the cells to migrate.This possibility is currently being examined.That basal migration in wounded cells was also inhibited by PA1 could be due to the inhibition of PAS released by the cells after wounding or formed from growth factors which is released on wounding ($27). EACA,a non-specific protease inhibitorantagonized tPA induced HWEcell migration in our assay. Some studies indicate that EACA acts by plasmin inhibition (8). Since we used a system that could not generate plasmin to any appreciable extent, we suggest that this inhibition is a result of EACA’s effect on the lysine-binding sites of tPA, as shown previously (28). In conclusion,we have demonstrated that tPA is capable of stimulating HUVEcell migration in wound assays. We suggest that tPA may play some role in EC migration, at least in large vessels,where it is produced,is readily secreted on stimulation and can bind to its cell surface receptor; requirements necessary for induction of migration (7). Such a role could be complimentary to that of uPA in the healing of large vessel lesions and deserves further studies.
ACKNOWLEDGMENT:
This study was supported by the Fondation pour la Recherche
Medicale for ALI.
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l&Montesano, R., Vassalli, J.D., Baird, A., Gullemin, R.& Grci,L.(1986) ProcNatl. Acad.Sci.USA 83,7297-7301. 19.Alessi,M.C.,DeClerck, J.P., DeMol, M., Nelles, L.& Collen,D.(1988). Eur.J. B&hem, 175.531-540. tO.Burk, R.R.(1973). Proc.Natl.Acad.Sci.USA. 70, 369-372. 21.Jaffe, E.A., Nachman, R.L., Becker, C.G.& Minick, C.R.(1973) J.Clin.Invest. 52, 2745-2756. 22.Hajjar, K.A., Harpel,P.A.,Jaffe, E.A.&.Nachman, R.L.(1986).J.Biol.Chem.261, 11656-l 1662. 23.Schleef, R.R.& Birdwell, CR. (1982). Exp.Cell.Res. 141, 503-508. 24.Levin, E.G.& !&tell, L.( 1988). J.Biol.Chem. 263, 9360-9365. 2S.Presta, M., Maier, J.A.M.& Ragnotti, G.(1989). J.Cell.Biol. 109, 1877-1884. 26.Saksela, O., Moscatelli, D.& Rifkin, D.B.(1987) J.Cell.Biol. 105,957-963. 27Plaumenhaft, R., Moscatelli, D.,Saksela, O.& Rifkin, D.B.(1989). J.Cell.Physiol. 140, 75-81. 2&Beebe,D. (1987). Thromb.Res. 46, 241-254.
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