BIOCHEMICAL

Vol. 181, No. 2, 1991 December

16, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 902-906

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) INDUCES PLASMINOGEN ACTIVATORS AND PLASMINOGEN ACTIVATOR INHIBITOR-l IN MICROVASCULAR ENDOTHELIAL CELLS M.S.Pepper,*N.Ferrara,

L.Orci and R.Montesano

Institute of Histology and Emb ology, Department of Morphology, University of Geneva Medical z! enter, 1211 Geneva 4, Switzerland *Department of Cardiovascular Research, Genentech, South San Francisco, California 94080 Received

October

29,

1991

SUMMARY Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI- mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 3Ong/ml. u-PA and t-PA mRNAs were increased 7.5 and g-fold respectively after 15 hours, and PAI- mRNA 4.5fold after 4 hours exposure to VEGF. At e uimolar concentrations (0.5nM), VEGF was a more potent inducer of t-PA mRNA than b s GF, while bFGF was a more otent inducer of u-PA and PAI- mRNAs. In addition, VEGF induced u-PA and PA! -1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAIreduction by VEGF in microvascular endothelial cells and are in accord with the l-iypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesls. 0 1991 Academic PESS, Inc.

Angiogenesis, the formation of new capillary blood vessels from pre-existing vessels, consists of a number of sequential events including endothelial cell migration and division which result in the formation of a capillary sprout. We and others have suggested that at least three elements of the angiogenic process may be mediated by extracellular proteolysis: degradation of the investing basement membrane of the parent vessel, invasion of the interstitial extracellular matrix by migrating endothelial cells, and capillary lumen formation (reviewed in 1 and 2). PAS are key mediators of these processes, and we have suggested that PA activity must be balanced by physiological PA inhibitors such as endothelial cell-derived PAT-1 for normal capillary morphogenesis (3,4; reviewed in 1). use& bFGF - basic fibroblast growth factor; BME cell - bovine microvascular endothelial cell; PA - plasminogen activator; PAI- PA inhibitor-l; SDS/PAGE - sodium dodecyl sul hate olaycrylamide gel electro horesis; t-PA - tissuetype PA; u-PA - urokinase-type IblJ A, GF - vascular endothelia P growth factor; VPF vascular permeability factor. 0006-291X/91 Copyright All rights

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

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Different polypeptide growth factors have been demonstrated to be angiogenic in vivo, the most completely characterized of which is bFGF (reviewed in 5). bFGF has been shown to stimulate a number of endothelial cell functions that are relevant to the process of angiogenesis, including endothelial cell migration and proliferation (reviewed in S), and the production of PAS (6, 7) and PAI-1 (8, 3). VEGF is a more recently described angiogenic factor (9-11) which is apparently an endothelial cell-specific mitogen (9, ll-13), in contrast to bFGF, which is mitogenic for a broad spectrum of cell types (reviewed in 5). In this study we have investigated the effects of VEGF on microvascular endothelial cell PA and PAT-1 production. MATERIALS

AND METHODS

Recombinant human VEGF (165-amino acid s ecies) was purified from transfected Chinese hamster ovary cells as previously described P14). The purity of the material was assessed by SDS/PAGE and by the presence of a single N&-terminal amino acid sequence. Levels of endotoxin were < 0.3IU/mg protein. BME cells were cultured as described (6) in gelatin-coated tissue culture dishes or flasks (Falcon Labware, Becton-Dickinson Company, Lincoln Park, NJ) in minimal essential medium, alpha-modification (alpha-MEM, Glbco AG, Basel, Switzerland), 15% heat inactivated donor calf serum (Flow Laboratories, Ayrshire, Scotland), penicillin (SOOIU/ml) and streptomycin (lOOpg/ml). For zymo raphic analysis, confluent monolayers of BME cells were washed twice with PBS, and vi GF was added at the indicated concentrations in serum-free alpha-MEM containing Trasylol (200KIU/ml) (Bayer-Pharma AG, Zurich, Switzerland). 15 hours later, cell extracts and culture supernatants were prepared, and analyzed zymographically as previously described (15). Total cellular RNA was prepared from confluent monolayers of BME cells exposed either to recombinant human bFGF (kindly provided by Dr.P.Sarmientos, Farmitalia Carlo Erba, Milan, Italy) or VEGF in complete BME medium. RNA preparation,. Northern blots., UV cross-hnking and methylene blue staining of filters, in vrtro transcription and hybndization were as previously described (3). cRNA probes were prepared from mouse uPA (16), human t-PA (17) and bovine PAI- (3) cDNAs. Autoradio raphs were scanned with a GenoScan laser scanner (Genofit, Geneva, Switzerland) at di 2 erent exposure times to avoid saturation. Results are expressed relative to control, non-treated cultures at the corresponding time point. RESULTS Zymographic analysis revealed the induction of both u-PA and t-PA activity by VEGF in a dose-dependent manner, with maximal induction at approximately 3Ong/ml (Figure 1). t-PA (complexed to PA&l) was detected principally in the culture supernatant of BME cells, while u-PA was predominantly cell associated (Figure 1; cf. ref 2). u-PA was distinguished from t-PA and t-PA/PA&l complex by zymographic analyses on the basis of inhibition of its catalytic activity by amiloride (18). t-PA/PA&l complex was characterized by Loskutoff et al. on the basis of its recognition by antibodies to either t-PA or PAI- by Western blot (19). u-PA and t-PA mRNAs were increased 7.5- and g-fold respectively after 15 hours, and PAI- mRNA 4.5fold after 4 hours exposure to VEGF (Figures 2 and 3). When 903

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4.PAfPAl.l -t.PA -wPA

-t-PA

c

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Fiure 1. Zymogra hit analysisof (A) serum-freetissueculture su ernatantsand (B) cell extracts from con!Iuent monolayersof BME cellsexposedto VEG i at the indicatedconcentrations for 15hours.t-PA (bound to PAL1 in culture supernatantsand free in cel1extracts) and uPA activity are both induced by VEGF in a dose-dependentmanner, with maximal induction at 30ng/ml.

compared to bFGF at equimolar concentrations (OSnM), VEGF was a more potent inducer of t-PA mRNA, while bFGF was a more potent inducer of u-PA and PAImRNAs (Figures 2 and 3). In addition, VEGF induced u-PA and PAL1 mRNAs with kinetics similar to those previously demonstrated for bFGF (Figures 2 and 3, and cf. ref 3). In response to bFGF and VEGF respectively, the ratio of u-PA:PAI-1 mRNA was 0.27 and 0.60 after 4 hours, and 2.29 and 2.70 after 15 hours. DISCUSSION VEGF, like bFGF, the most completely characterized angiogenic factor to date, is angiogenic both in vivo (9-11) and in vitro (Pepper et al., in preparation). However, VEGF differs from bFGF in a number of important respects. Firstly, VEGF appears to be an endothelial cell specific mitogen (9, ll-13), while bFGF is mitogenic for a broad spectrum of cell types (reviewed in 5). Secondly, VEGF is a secreted protein (10,20-22), while bFGF lacks a signal sequence and does not enter the classical secretory pathway (reviewed in 5). Thirdly, VPF, a protein with a high degree of sequence similarity to VEGF (21), induces vascular permeability (23, 9), while bFGF has not been reported to do so. Here we demonstrate an additional difference between these two angiogenic factors: while bFGF mainly induces u-PA, VEGF induces both u-PA and t-PA. Both factors induce PAI-1. These results also suggest that bFGF and VEGF are acting via different regulatory pathways. u-PA has been implicated in processes of cell migration and tissue remodelling, while t-PA is believed to be involved mainly in intravascular thrombolysis (reviewed in 2). 904

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15 hrs - 28s u-PA “7 20

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Fieure 2. u-PA, t-PA and PAl-1 mRNA induction by bFGF and VEGF. Confluent monola ers of BME cells were exposedto equimolar (OSnM) concentrationsof bFGF (9ngPml) or VEGF (22Sng/ml), and total cellular RNA extracted after 4 or 15 hours.Northern blots were hybridized with 32P-labelledcRNA probesas describedin Materials and Methods. Methylene blue staining revealed uniform loading of RNAs and intact 28s and 18s ribosomalRNAs after transfer and UV cross-linkingto nylon filters (bottom panel of the figure). Figure 3, Quantitation by densitometricscanningof u-PA, t-PA and PAL1 mRNA induction by bFGF and VEGF, asshownin Figure2.

Using the ratio of u-PAzPAI-1 mRNAs as an indicator of potential proteolytic activity, we have proposed that an appropriate balance between proteases and protease inhibitors is a necessary requirement for normal capillary morphogenesis (3). Here we have shown that the ratio of u-PA:PAI-1 mRNAs is similar for bFGF and VEGF. The positive increase in the proteolytic balance may mediate the extracellular proteolytic events which are required for angiogenesis (3). Finally, the role of VEGF-induced t-PA production is at present unknown. Whether it is required for intravascular thrombolysis or other functions of VEGF such as increased vascular permeability, remains to be determined.

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ACKNOWLEDGMENTS We are grateful to Dr. J.-D.Vassalli for his useful comments and advice, Drs. M.B.Furie and S.C.Silverstein for providing the BME cells, Drs. D.Belin and W.Schleuning for mouse u-PA and human t-PA cDNA probes, respectively, and Dr. P.Sarmientos for recombinant human bFGF. Excellent technical assistance was provided by C.DiSanza and M.Guisolan, and photographic work was done by J.-P.Gerber. This work was supported by a grant from the Swiss National Science Foundation (no. 31-26625.89) and a grant in aid from the Sir Jules Thorn Charitable Overseas Trust. REFERENCES 1. Pepper, M.S., and Montesano, R. (1991) Cell Diff. Dev. 32,319-328. Moscatelli, D., and Rifkin, D.B. (1988) Biochim. Biophys. Acta 948,67-85. S: Pepper, M.S., Belin, D., Montesano, R., Orci, L., and Vassalli, J.-D. (1990) J. Cell Biol. 111,743-755. 4. Montesano, R., Pepper, M.S., Miihle-Steinlen, U., Risau, W., Wagner, E.F., and Orci, L. (1990) Cell 62,435-445. Klagsbrun, M., and D’Amore, P.A. (1991) Ann. Rev. Physiol. 53,217-239. 2: Montesano, R, Vassalli, J.-D., Baird, A., Guillemin, R., and Orcr, L. (1986) Proc. Natl. Acad. Sci. USA 83,7297-7301. 7. Moscatelli, D., Presta, M., and Rifkin, D.B. (1986) Proc. Natl. Acad. Sci. USA 83,209l -2095. 8. Saksela, O., Moscatelli, D., and Rifkin, D.B. (1987) J. Cell Biol. 105,957-963. 9. Connolly, D.T., Heuvelman, D.M., Nelson, R., Olander, J.V., Eppley, B.L., Delfino, J.J., Siegel, N.R., Leimgruber, R.M., and Feder, J. (1989) J. Clin. Invest. 84, 1470-1478. 10. Leung, D.W., Cachianes, G., Kuang, W.-J., Goeddel, D.V., and Ferrara, N. (1989) Science 246, 1309-1312. Plouet, J., Schilling, J., and Gospodarowicz, D. (1989) EMBO J. 8,3801-3806. 2 Ferrara, N., and Henzel, W.J. (1989) Biochem. Biophys. Res. Commun. 161,851-858. 13: Gos odarowicz, D., Abraham, J.A., and Schilling, J. (1989) Proc. Natl. Acad. Sci. USA 86. 7 311-7215. 14. Ferrara, N., Leung, D.W., Cachianes, G., Winer, J., and Henzel, W.J. (1991) Methods Enzymol. 198,391-404. 15. Vassalli, J.-D., Dayer, J.-M., Wohlwend, A., and Belin, D. (1984) J. Exp. Med. 159, 1653-1668. 16. Belin, D., Vassalli, J.-D., Combepine, C., Godeau, F., Nagamine, Y., Reich, E., Kocher, H.P.. and Duvoisin. R.M. (1985) Eur. J. Biochem. 148: 225-232. 17. Fisher ,a--->’ R., Waller, _ -_ E.K, Grossi; G., Thompson, D., Tizard, R., and Schleuning, W. (1YW) J. NoI. Chem. Zb(3,11223-l 1230. 18. Pepper, M.S., Vassalli, J.-D., Montesano, R., and Orci L. (1987) J. Cell Biol. 105,25352541. 19. Loskutoff, D.J., Ny, T., Sawdey, M., and Lawrence, D. (1986) J. Cell. Biochem. 32,273280. 20. Conn, G., Soderma, D.D., Schaffer, M.-T., Wile, M., Hatcher, V.B., and Thomas, K.A. (1990) Proc. Natl. Acad. Sci. USA 87,1323-1327. 21. Keck, P.V., Hauser, S.D., Krivi, G., Warren, T., Feder, J., and Connolly, D.T. (1989) Science 246, 1309-1312. 22. Tischer, E., Gos odarowicz, D., Mitchell, R., Silva, M., Schilling, J., Lau, K., Crisp, T., Fiddes, J.C., an cf Abraham, J.A. (1989) Biochem. Biophys. Res. Commun 165,11981206. 23. Senger, D.R., Galli, S.J., Dvorak, A.M., Perruzzi, C.A., Harvey, V.S., and Dvorak, H.F. (1983) Science 219,983-985.

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Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic fac...
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