Tumor cells secrete an Angiogenic factor that stimulates basic fibroblast growth factor and...

JOURNAL OF CELLULAR PHYSIOLOGY 161:l-14 (1994)

Tumor Cells Secrete an Angiogenic Factor That Stimulates Basic Fibroblast Growth

Factor and Urokinase Expression in Vascular Endothelial Cells

FIORENZO A. PEVERALI, STEFAN0 J. MANDRIOTA, PAOLO CIANA, ROSARIA MARELLI, PAUL QUAX, DANIEL B. RIFKIN, C l U L l A N O DELLA VALLE,

AND P A 0 1 0 MIGNATTI * Dipartimento di Cenetica e Microbiologia, Universita di Pavia, 27 100 Pavia, Italy; (F.A. P.,

S.I.M., P.C., R.M., C.D.V., P.M.); TNO-IVVO Caubius Laboratory, 2300 AK Leiden, The Netherlands (P.Q.); Department of Cell Biology and Kaplan Cancer Center, New York

University Medical Center, and the Raymond and Beverly Sackler Laboratories, New York, New York 100 16 (D.B. R.)

Culture medium conditioned by human SK-Hepl hepatoma cells or mouse S180 sarcoma cells rapidly up-regulates endothelial cell expression of basic fibroblast growth factor (bFGF) and induces formation of capillary-like structures by vascular endothelial cells grown on three-dimensional fibrin gels (in vitro angiogenesis). Incubation of endothelial cells with the tumor cell-conditioned media also results in increased expression of urokinase plasminogen activator (uPA), a key component of the proteolytic system required for cell invasion and capillary formation. Although the tumor cell-conditioned media contain no bFGF, addition of anti- recombinant bFGF IgG abolishes the up-regulation of uPA and blocks in vitro angiogenesis. This indicates that both the increase in uPA production and formation of capillary-like structures are mediated by endogenous bFGF expressed by the endothelial cells. Both the bFGF/uPA-inducing activity and the angiogenic activity of SK-Hepl cell-conditioned medium copurify with a relatively acid- resistant peptide that has moderate affinity for heparin and M, < 18 kDa > 3.5 kDa. Known cytokines with similar biochemical features do not possess the same biological activity. These findings indicate that angiogenesis can be mediated by endothelial cell bFGF through an autocrine mechanism and that the bFGF-inducing peptide may represent a novel tumor-derived angiogenic factor that modulates in endothelial cells the concerted expression of cytokines and proteolytic enzymes required for capillary formation. o 1994 Wiiey-Liss, tnc.

The growth of solid tumors, as well as their ability to metastasize, are dependent upon the process of vascu- larization (angiogenesis) (Folkman, 1990; Folkman et al., 1989; Weidner et al., 1991). In the presence of a small tumor cell nidus, endothelial cells within an adja- cent venule locally degrade the vascular basement membrane and migrate into the interstitial matrix to form a capillary network, while cell proliferation occurs at the base of the newly forming capillary (Ausprunk et al., 1975; Ausprunk and Folkman, 1977; Folkman, 1986). These functions are mediated in endothelial cells by a number of cytokines with angiogenic activity.

Among these cytokines, basic fibroblast growth factor (bFGF) is one of the more potent angiogenesis in- ducers in a variety of in vivo assays (Basilico and Mos- catelli, 1992; Folkman and Klagsbrun, 1987; Shing et al., 1985). In vitro bFGF stimulates endothelial cell proliferation (Gospodarowicz et al., 1985; Schweigerer et al., 1987), migration (Sato and Rifkin, 19881, formation of patent capillary-like structures in collagen or fibrin gels (Montesano et al., 1986), and production of 0 1994 WILEY-LISS, INC.

proteinases, including urokinase-type plasminogen activator (uPA), uPA receptor, and collagenase (Mignatti et al., 1991a; Moscatelli et al., 1986b; Pepper et al., 1990). These enzymes are required for ECM degradation during cell invasion (Mignatti and Rifkin, 1993; Mignatti et al., 1986, 1989). Although bFGF lacks a secretory signal peptide (Abraham et al., 1986), it modulates several cell functions in an autocrine manner (Mignatti et al., 1991b; Sato and Rifkin, 1988; Sato et al., 1991). Significant amounts of bFGF are associated with the ECM of in vitro cell cultures (Bashkin et al., 1989; Flaumenhaft et al., 1989; Rogelj et al., 1989; Vlo- davski et al., 1987). In vivo bFGF has been located in the basement membranes of blood vessels (DiMario et

Received July 20,1993; accepted March 7, 1994. "Address correspondence to Dr. Paolo Mignatti, Dipartimento di Genetica e Microbiologia, Universita di Pavia, 27100 Pavia, Italy.

PEVERALI ET AL 2

al., 1989; Folkman et al., 1988), mainly within the basal lamina of branching capillaries (Cordon et al., 1990) and in the capillary endothelial cells of some tumors (Schulze-Osthoff et al., 1990).

The interactions of uPA and plasmin with a number of components of the extracellular microenvironment have relevant effects on endothelial cells. 1) Plasmin degrades most ECM components, either directly or indirectly through the activation of the zymogen forms of matrix metalloproteinases (Mignatti and Rifkin, 1993). The degradation of the vascular basal lamina affords endothelial cell invasion into interstitial stroma; in addition, ECM degradation results in the mobilization of growth factors, including bFGF, bound to proteogly- cans in the matrix (Saksela and Rifkin, 1990; Flaumen- haft et al., 1990). 2) Plasmin efficiently activates latent transforming growth factor beta (TGFP) (Lyons et al., 1990; Flaumenhaft et al., 1992; Sat0 and Rifkin, 1989; Sato et al., 1990). Active TGFP down-regulates uPA expression and stimulates the synthesis of type 1 plasminogen activator inhibitor (PAI-11, the major PA inhibitor produced by endothelial cells (Pepper et al., 1990; Saksela et al., 1987). The resulting blockade of plasmin formation also turns off the plasmin-mediated activation of TGFP, generating a self-regulatory mechanism for the control of plasmin activity (Flaumenhaft et al., 1992; Sat0 et al., 1990). Thus, the uPA-plasmin system plays a key role in the modulation of both extracellular proteolytic activity and growth factor effects on endothelial cells during angiogenesis.

However, although the role of bFGF and uPA in angiogenesis is supported by a considerable body of experimental evidence in vitro and in vivo, i t contrasts with the finding that endothelial cells in vivo remain quies- cent for many years in spite of the presence of significant amounts of bFGF in vascular basement membranes (Cordon et al., 1990; Vlodavski et al., 1990, 1991). Moreover, in spite of the constant presence of bFGF in the ECM, uPA expression in forming capillaries in vivo appears to be temporally restricted (Bach- arach et al., 1992). In the light of these observations we speculated that the initial event that triggers angiogenesis may consist of an increased expression of bFGF by vascular endothelial cells, rather than of a paracrine effect of bFGF derived from other cells or the ECM. The finding of high levels of bFGF in the capillary endothelial cells of some tumors (Schulze-Osthoff et al., 1990) suggests that this mechanism might be prominent during tumor angiogenesis. To test this hypothesis we characterized t,he effect of medium conditioned by tumor cells on bFGF expression by vascular endothelial cells. Among the tumor cell lines tested, SK-Hepl human hepatoma cells and S180 mouse sarcoma cells were of particular interest. S180 cells do not express bFGF (Moscatelli et al., 1986a) but secrete an angiogenic activity that promotes the growth of endothelial cells in vitro, and their conditioned medium has long been used as a growth supplement for endothelial cell cultures (Folkman et al., 1979). SK-Hepl cells produce high levels of bFGF, although they do not secrete it into the culture medium (Moscatelli et al., 1986a; Sternfield et al., 1988). We hypothesized that the high expression of bFGF in these cells might result from an autocrine stimulation by a soluble factor.

MATERIALS AND METHODS Reagents

For mRNA analysis the following cDNA fragments were used as probes: an 800 bp EcoRI fragment from human bFGF cDNA, kindly provided by Dr. J.A. Abra- ham (California Biotechnology Inc., Mountain View, CA); a 1,050 bp EcoRI fragment from human TGFPl cDNA, kindly provided by Dr. R. Derynck (Depart- ments of Growth and Development and of Anatomy, University of California, San Francisco, CA); a 3 Kb EcoRI fragment from human PAI-1 cDNA, a 1.9 Kb BglII fragment from human tPA cDNA, and a 400 bp EcoRI fragment from human uPA cDNA, kindly provided by Dr. J.-F. Cajot (Unit of Experimental Pathol- ogy, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland). Human recombinant bFGF (rbFGF) was a kind gift from Synergen (Boulder, CO). Rabbit anti-human rbFGF IgG was purified by rbFGF- Affi-Gel affinity chromatography and tested for neutralizing activity as described (Mignatti et al., 1991b). The IgG not retained by the rbFGF-Affi-Gel column was used as control. The affinity-purified antibody im- munoprecipitates bFGF from extracts of NIH 3T3 cells transfected with human bFGF cDNA (authors’ unpublished results; Bikfalvi et al., manuscript in preparation). Recombinant human macrophage inflammatory protein (MIP) 2a and 2b and recombinant mouse MIPla and MIPlb, as well as specific antibodies to these cytokines, were a generous gift from Dr. A. Cerami (Rock- efeller University, New York, NY). Vascular endothelial cell growth factor (VEGF) was kindly provided by Dr. D. Gospodarowicz (University of California, San Francisco). Heparin-Sepharose CL-6B was purchased from Pharmacia LKB (Upsala, Sweden), protein A-Sepharose CL-4B from Sigma (St. Louis, MO), soybean trypsin inhibitor (STI) (57 Uimg) from Serva Fein- biochemica GMBH (Heidelberg, Germany), and trypsin from Gibco Laboratories (Gaithersburg, MD).

Cells and media Primary cultures of bovine capillary endothelial

(BCE) cells were obtained from the adrenal glands of yearling calves and tested for von Willebrand factor- related antigens as described (Folkman et al., 1979). BCE cells were grown in gelatin-coated petri dishes in alpha minimal essential medium (aMEM) (Gibco) supplemented with 5% Donor Calf Serum (DCS) (Flow Lab- oratories, McLean, Virginia) and 2 mM L-glutamine (Gibco). Bovine aortic endothelial (BAE) cells were obtained from the adult aortic arch of yearling calves as described (Schwartz, 1978). The cells were grown in gelatin-coated dishes in aMEM containing 10% DCS and 2 mM L-glutamine. BCE cl. 1 and BCE el. 113 cells were used at the fifth passage in culture, BCE 8A2 cells between passage 8 and 18, and BAE cells a t passage 10. Cells of the fetal bovine aortic endothelial cell line GM 7373 were kindly provided by Dr. M. Presta (Universita di Brescia, Italy). The cells were grown in Dulbecco’s modified MEM (DMEM) (Gibco) containing 20% fetal calf serum (FCS) (Flow Laboratories), 2% essential amino acids (Gibco), 2% non-essential amino acids (Gibco), 2% vitamins (Gibco), and 2 mM L-glutamine. Human fibroblasts were obtained from adult skin and grown in DMEM containing 10% FCS and 2 mM L-glu-

3 INDUCTION OF ENDOTHELIAL CELL bFGF AND UROKINASE

tamine. Human HT1080 fibrosarcoma cells, human SK-Hepl hepatoma cells, and mouse S180 sarcoma cells were grown in DMEM containing 10% FCS and 2 mM L-glutamine. Human U237 glioma cells were kindly provided by Dr. G. Damiani (IST, Genova, Italy). The cells were grown in RPMI medium supplemented with 15% FCS.

Preparation of cell-conditioned media Confluent cultures of primary fibroblasts or tumor

cells in 150 cm2 culture flasks were washed twice with phosphate buffer saline (PBS) and incubated for 18 h in the presence of 10 ml of serum-free DMEM. The culture supernatant was centrifuged at 9,800g for 15 min and either tested immediately or stored at -20°C. Condi- tioned media were supplemented with 1% DCS before assaying for bFGF- or uPA-inducing activity.

Assay for bFGF-inducing activity Confluent endothelial cell cultures were split 1:4 and

incubated at 37°C in growth medium. As soon as the cells were confluent, the medium was changed, and the cultures were incubated at 37°C for 5 days with no medium change. The cells were then washed twice with PBS and incubated with the conditioned media at 37°C for the indicated times. Incubation was stopped by put- ting the culture plates on ice for RNA extraction.

Northern blotting Total RNA was extracted from cells as described

(Auffray and Rougeon, 1980). Total RNA (20 pg) was run in a 1.0% formaldehyde-agarose gel, transferred to a nitrocellulose membrane (Hybond C-Extra; Amer- sham, England), prehybridized, and hybridized to ran- dom priming-[32P]dCTP-labeled cDNA probes (approximately lo9 cpm/pg) as described (Sambrook et al., 1989). After hybridization, the membranes were washed twice in 3 x SSC a t room temperature, twice in I X SSC a t 42"C, and twice under different stringent conditions ( 0 . 2 ~ SSC, 50°C for bFGF, uPA, PAI-1, and tPA probes; 0.1 x SSC, 60°C for the TGFPl probe). The membranes were exposed to Agfa films a t -80°C.

Densitometric analysis of mRNA levels The autoradiographic signals obtained by Northern

blotting with 32P-labeled cDNA probes were quantitated by scanning densitometry both with a CAMAG TLCII optical scanner and with a Hewlett Packard ScanJet IIC interfaced to a Macintosh LCIII computer with DeskScan I1 Image software. The two methods gave comparable results. For each sample the densitometric value obtained with bFGF, TGFP1, uPA, or PAI-1 cDNA probes was normalized to the corresponding value obtained with the GAPDH probe to adjust for variability in the loading and blotting of the RNA samples. To check the linearity of the densitometric analysis, increasing amounts of 1251-~PA (0.15 pCi/pMole) were run in a 10% SDS-polyacrylamide gel. The gel was exposed to autoradiographic films for different times, and autoradiographic bands were analyzed as described above. The response was linear within the range of intensity of the bands shown in the figures.

Assay for PA-inducing activity Confluent endothelial cells in 24-well microculture

plates (Costar, Cambridge, MA) were incubated at 37°C for 5 days with no medium change. The cells were then washed twice with PBS and incubated with the conditioned media a t 37°C for the indicated times. At the end of the incubation, the cultures were washed three times with cold PBS and lysed with 0.5% Triton X-100 in Tris-HC1 0.1 M, pH 8.1. Cell extract protein (1-10 pg) was tested for PA activity by the lZ51-fibrin assay (Gross e t al., 1982) or by fibrin zymography (Granelli- Piperno and Reich, 1978).

Neutralization of uPA-inducing activity with anti-rbFGF antibody

Two-milliliter aliquots of SK-Hepl or S180 cell-conditioned medium was incubated at 4°C for 1 h in the presence of either 10 pg/ml of irrelevant IgG or affinity- purified anti-rbFGF IgG or, as a control, 20 p1 of PBS. At the end of the incubation each sample was split into two 1 ml aliquots. Protein A-Sepharose (50 11.1) or 50 pl of PBS was added to parallel aliquots, and incubation was continued for 1 h at 4°C with mild agitation. Con- trol medium was supplemented with the same volumes of PBS and incubated at 4°C for the same time as the conditioned media. The samples were centrifuged for 10 seconds in an Eppendorf centrifuge, and the supernatants were tested for PA-inducing activity on BCE cells as described above.

Characterization of bFGF/uPA-inducing activity Dialysis. Two-milliliter aliquots of SK-Hepl cell-

conditioned medium were put into Centricon tubes (Amicon, Beverly, MA) with 3.5 kDa or 10.0 kDa M, cutoff membranes and centrifuged at 5,OOOg for 30 min in a Sorvall centrifuge at 4°C. The retentate was diluted to the original volume with serum-free DMEM. Retentate and dialysate were supplemented with 1% DCS and tested for bFGF- and uPA-inducing activity. As a control, 1 pg of rbFGF in 2 ml of DMEM was dialyzed in Centricon tubes with 10.0 kDa M, cutoff membranes under the same conditions. Aliquots (10 pl) of the retentate and dialysate were diluted in 1 ml of DMEM containing 1% DCS and tested for uPA-inducing activity after 16 h incubation with BCE cells.

Trypsin digestion. Trypsin was added to SK-Hepl cell-conditioned medium to a final concentration of 0.5 mg/ml. After incubation a t 37°C for 30 min, 5 mg/ml of soybean trypsin inhibitor was added, and the medium was incubated in ice until tested.

Heparin-Sepharose chromatography. SK-Hepl cellkonditioned medium was dialyzed overnight in dialysis tubes with 3.5 kDa M, cutoff membranes vs. Tris- HCl50 mM, NaCl50 mM, 0.01% Tween 20, pH 7.0. The dialyzed medium was loaded onto a 1 ml heparin- Sepharose column equilibrated with the same buffer, a t a flow rate of 10 ml/h at 4°C. The column was washed with equilibration buffer until no O.D.,,, was mea- sured. Stepwise elution was carried out with NaCl 0.6 M, 1.0 M, and 2.5 M in the column buffer a t a flow rate of 50 ml/h. Alternatively, the column was eluted with a continuous NaCl gradient from 0.1-3.0 M. In both cases 0.5 ml fractions were collected. In the case of the step-

4

bFGF

GAPDH

A

PEVERALI ET AL.

B C D

- 28s

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H U M A N U 237 BCE BCE BCE BAE GM cl. 8A2 cl. 1 cl.113

Fig. 1. Basic FGF expression in vascular endothelial cells, fibroblasts, and glioma cells incubated with normal fibroblast- or tumor cell-conditioned medium. Northern blotting analysis of bFGF mRNA in (A) BCE 8A2 cells incubated for 4 h with control medium (CON- TROL), SK-Hepl cell-conditioned medium (SK-HEPl), human fibroblast-conditioned medium (H. FIBR.), S180 cell-conditioned medium (S180), or HT1080 cell-conditioned medium (HT1080); in (B) BCE cl. 1 and BCE cl. 1/3 cells, bovine aortic endothelial (BAE) cells, and transformed fetal bovine aortic endothelial cells (GM7373), incubated for 4 h with control medium (CONTROL) or SK-Hepl cell-conditioned medium (SK-HEP1); in (C) human adult skin fibroblasts (HUMAN FI- BROBLASTS) grown for 2 days in serum-free medium (NO SERUM)

wise elution the O.D.,so peaks eluted with the different NaCl concentrations were pooled and diluted with DMEM to 50 ml (original sample volume). This material was tested for bFGF-inducing activity. In the case of the gradient elution, 10 p1 of each fraction was diluted in 1 ml of DMEM containing 1% DCS and assayed for uPA-inducing activity. As a control, 1 pg of rbFGF in 1 ml of DMEM was loaded onto the same column and eluted with a continuous NaCl gradient from 0.1-3.0 M. Aliquots (10 pl) of the fractions collected were diluted in 1 ml of DMEM containing 1% DCS and tested for uPA-inducing activity after 16 h incubation with BCE cells.

In vitro angiogenesis assay Fibrin gels were reconstituted from bovine fibrino-

gen as described (Montesano et al., 1987). Briefly, bovine fibrinogen (Sigma) was dissolved in calcium- and magnesium-free PBS at a concentration of 2.5 mg/ml. Thrombin (Topostasin; Roche, Basel, Switzerland) was

7373 F IBROBLASTS

or incubated for 4 h with control medium containing 1% DCS (1% CS), or SK-Hepl cell-conditioned medium supplemented with 1% DCS (SK-HEPl), or control medium supplemented with 10% FCS (10% FCS); and in (D) human glioma cells (U237) incubated for 4 h with control medium (CONTROL) or SK-Hepl cell-conditioned medium (SK-HEP1). RNA extraction, blotting, and hybridization to a ."P-labeled cDNA probe to human bFGF were carried out as described in Materials and Methods. GAPDH mRNA is shown as a control. The experiments with endothelial cells were repeated four times, those with fibroblasts and U237 cells twice. Similar results were obtained in all the experiments.

dissolved in tenfold concentrated DMEM, pH 7.2, a t a concentration of 25 U/ml. Clotting was initiated by mixing 50 ml of the thrombin with 450 ml of the fibrinogen solution in 16 mm tissue culture wells (Nunclon; Roskilde, Denmark), and the gels were allowed to poly- merize at room temperture for 3 min. BCE or BAE cells were seeded on fibrin gels at a density of 2.5 x lo5 cells/well. After the cells had spread on the fibrin gel, the culture medium was replaced with either SK-Hepl cellkonditioned medium or medium supplemented with 100 pl/ml of the samples to be tested. Growth medium containing 100 pliml of Tris-HC1 50 mM pH 7.4, NaCl50 mM, Tween 20 0.01% was used as control. The medium was replaced every day. After 4 days of incubation at 37°C the cultures were photographed in phase contrast using a Leitz inverted photomicroscope. To remove the layer of non-invasive endothelial cells from the gel surface, the cultures were treated for 5 min a t 37°C with bacterial collagenase (type 1A; Sigma) 15 mg/ml in PBS containing 5 mg/ml of BSA. The treatment was stopped by removing the collagenase solution

INDUCTION OF ENDOTHELIAL CELL bFGF AND UROKINASE 5

lSm 30" 60" 2h 4 h 8h 16h --- ----

bFGF

TGF /3l

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Fig. 2. Kinetics of bFGF and TGFpl expression in BCE cells incubated with tumor cell-conditioned medium. Northern blotting analysis of bFGF and TGFpl mRNAs in BCE 8A2 cells incubated for the indicated times with control medium (CONTROL) or with S180 cell- conditioned medium (S180) or SK-Hepl cell-conditioned medium (SK-

HEP1). RNA extraction, blotting, and hybridization to 32P-labeled probes were carried out as described in Materials and Methods. GAPDH mRNA is shown as a control. This experiment was repeated three times with similar results.

and washing the cells repeatedly with calcium- and magnesium-free PBS.

RESULTS bFGF and uPA expression in vascular endothelial cells incubated with tumor

cell-conditioned medium Conditioned media from primary normal fibroblasts

or tumor cell lines were tested for their ability to stimulate basic fibroblast growth factor (bFGF) expression in capillary endothelial (BCE) cells. By Northern blotting (Fig. 1A) both the 7.0 Kb and the 3.7 Kb bFGF transcripts were more abundant in cells incubated with human SK-Hepl hepatoma or mouse ,5180 sarcoma cell-conditioned medium than in cells grown in control medium. In contrast, medium conditioned by primary human fibroblasts or HT1080 human fibrosarcoma cells did not induce any increase in the level of bFGF mRNA.

The effect of SK-Hepl cell-conditioned medium was tested on several different clones of capillary endothelial cells (BCE 8A2, BCE cl. 1, and BCE cl. 1/31, on primary bovine aortic endothelial (BAE) cells and transformed fetal bovine aortic cells (GM7373), and on human fibroblasts and U237 glioma cells. These cells express different basal levels of bFGF mRNAs (Fig. 1B). In all the endothelial cells, SK-Hepl cell-conditioned medium increased the level of bFGF mRNAs. The stimulatory effect inversely correlated with the basal level of bFGF mRNA expressed by the cells. The conditioned medium also increased the level of bFGF

mRNA in serum-starved primary human fibroblasts. This effect was comparable to that obtained by addition of 10% FCS (Fig. 1C). In contrast, SK-Hepl cell-conditioned medium had no effect on the level of bFGF transcripts of human U237 glioma cells (Fig. 1D).

Time-course experiments (Fig. 2) showed that tumor cell-conditioned medium induced an increase in bFGF mRNA level, relative to cells incubated in control medium, as early as 15 min after medium change. High levels of bFGF transcripts were maintained up to 8 h of incubation. By scanning densitometry (Fig. 3) the level of the 7.0 Kb bFGF transcript increased up to fourfold in cells incubated with S180 cell-conditioned medium for 30 min and 2.2-fold in cells incubated with SK-Hepl cell-conditioned medium for 30 min, relative to cells incubated in control medium for the same time. The level of the 3.7 Kb transcript increased up to 3.5-fold in cells incubated with S180 cell-conditioned medium for 2 h, and threefold in cells incubated with SK-Hepl cell- conditioned medium for 4 h.

We also characterized the effect of S180 and SK-Hepl cell-conditioned media on BCE cell expression of other genes involved in cell proliferation andlor angiogenesis. A very slight increase in C-myc mRNA could be seen only in cells incubated with the conditioned media for 1 h (data not shown). No expression of K-fgfhst could be seen either in control cells or in cells treated with the conditioned media, whereas the levels of TGFa, c-sis, and PDGF receptor mRNAs did not show significant differences between control cells and cells treated with the conditioned media (data not shown). In contrast, in

6 PEVERALI ET AL.

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Fig. 3. Densitometric analysis of bFGF mRNA expression in BCE cells incubated with tumor cell-conditioned medium for different times. The 7.0 Kb and 3.7 Kb bFGF transcripts of BCE cells incubated with control medium (-O-), S180 cell-conditioned medium (A-), or SK-Hepl cell-conditioned medium (-1 were quantitated by scanning densitometry of autoradiographic films, including the one shown in Fig. 2, as described in Materials and Methods. For each point the signal obtained with the bFGF cDNA probe was normalized to the densitometric reading of the corresponding GAPDH transcript. The data are presented as fold increase relative to the silver grain density of the band corresponding to the 7.0 Kb or 3.7 Kb bFGF transcript of BCE cells incubated with control medium for 15 min. Each point represents mean & range of values obtained by the densitometric analysis of autoradiographic films from three separate experiments.

BCE cells incubated with SK-Hepl or S180 cell-conditioned medium, the level of TGFPl mRNA increased at 2 h and remained significantly higher than in control cells up to at least 16 h of incubation (Fig. 2). Scanning densitometry showed that TGFPl mRNA increased up to fivefold in cells treated with S180 cell-conditioned medium for 16 h and threefold in cells incubated with SK-Hepl cell-conditioned medium for 8 h, relative to cells incubated with control medium for the same time (data not shown).

Basic FGF strongly up-regulates plasminogen activator (PA) expression in vascular endothelial cells (Mignatti et al., 1991a; Moscatelli e t al., 198613; Pepper e t al., 1990; Saksela et al., 1987). Therefore, we investi- gated if the changes in bFGF expression induced in BCE cells by the tumor cell-conditioned media resulted also in variations of uPA or tissue-type PA (tPA). By Northern blotting (Fig. 4), four- to fivefold increases in uPA mRNA could be detected in BCE cells incubated with SK-Hepl or S180 cell-conditioned medium for 4 h. Interestingly, PAI-1 mRNA levels were also dramati- cally increased in cells treated with the conditioned media for the same time. In contrast, no tPA transcripts

n c z C O Y - w m m

uPA

PAI-1

- 1 8 s

GAPDH

- 1 8 s

Fig. 4. uPA and PAI-1 expression in endothelial cells incubated with tumor cell-conditioned medium. Northern blotting analysis of uPA and PAI-1 mRNAs in BCE 8A2 cells incubated for 4 h with control medium (Control) or SK-Hepl cell-conditioned medium (SK-Hepl) or S180 cell-conditioned medium (S180). RNA extraction, blotting, and hybridization to "'P-labeled probes were carried out as described in Materials and Methods. GAPDH mRNA is shown as a control. This experiment was repeated three times. Similar results were obtained with BCE cl. 1, BCE cl. 113, BAE, and GMi'373 cells.

were detected (data not shown). Fibrin zymography of BCE cell extracts (Fig. 5A) revealed one major band with M, 40-45 KDa, corresponding to the expected M, of bovine uPA (Saksela et al., 1987). In BCE cells incubated with SK-Hepl or S180 cell-conditioned medium, this band was larger and more intense than in cells incubated with control medium. No high-M, bands, rep- resenting tPA or uPA-PA1 complexes, were present either in control BCE cells or in cells treated with the conditioned media. The PA activity of BCE 8A2 cells was completely neutralized by antibody to bovine uPA but not by neutralizing anti-human uPA or anti-human tPA antibody; immunoprecipitation with anti-bovine uPA and anti-human tPA antibodies showed that all the PA activity was associated with uPA (data not shown). These results showed that the increase in PA activity of BCE cells incubated with tumor cell-conditioned medium was mediated by increased uPA expression. By the '251-fibrin assay the uPA activity of BCE cells incubated with SK-Hepl cell-conditioned medium increased rapidly and peaked a t 4 h; the PA activity of cells incubated with S180 cell-conditioned medium increased after 12 h and levelled off between 16 and 32 h of incubation (Fig. 5B). Similar results were obtained with BAE and GM7373 cells (data not shown). With both conditioned media the time course of uPA induction was significantly different from that of BCE cells incubated with a saturating concentration (550 pM) of rbFGF (Fig. 5B, dotted line). Higher or lower bFGF concentrations did not change the time course of uPA induction (data not shown), suggesting that the in-


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Fig. 5. Induction of PA activity in BCE cells by SK-Hepl and S180 cell-conditioned medium. A Fibrin zymography. Lane 1: SK-Hepl cell-conditioned medium. Lane 2: S180 cell-conditioned medium. Lanes 3-5 Triton X-100 extracts of BCE cells incubated with control medium (3) or SK-Hepl cell-conditioned medium (5) for 4 h or with S180 cell-conditioned medium (4) for 28 h. Lane 6 Pure human uPA. Conditioned medium (50 p1) or 10 pg of cell extract protein were run in a 10% SDS-polyacrylamide gel before fibrin zymography. This experiment was repeated five times. Similar results were obtained with BAE and GM7373 cells. B BCE 8A2 cells were incubated for the indicated times with control medium (-=-I, S180 cell-conditioned medium (-+-I, SK-Hepl cell-conditioned medium ( 2 - 1 or control medium containing 10 ng/ml of rbFGF (dot.ted line). Cell extracts were tested for PA activity by the lZ5I-fibrin assay as described in Materials and Methods. This experiment was repeated seven times; similar results were also obtained with BAE and 7373 cells. Each point represents mean ? range of variation in duplicate samples from a representative experiment.

crease in PA activity of BCE cells incubated with SK- Hepl or S180 cell-conditioned medium was not mediated by exogenous bFGF derived from the tumor cells.

S180 cells do not express bFGF; on the contrary, SK- Hepl cells produce high levels of bFGF, but the growth factor is not detectable in the culture supernatant (Mos- catelli et al., 1986a; Sternfield et al., 1988). By North- ern blotting we found high levels of bFGF mRNA in SK-Hepl cells but no detectable bFGF transcripts in S180 cells (data not shown). To examine whether the increase in PA activity of BCE cells incubated with the tumor cell-conditioned media was mediated by an increased bFGF expression in the endothelial cells or by bFGF present in the conditioned media, BCE cells were incubated with SK-Hepl or S180 cell-conditioned medium, in the presence or in the absence of 10 pg/ml of neutralizing affinity-purified anti-rbFGF IgG (Mig-

natti et al., 1991b) or irrelevant IgG. As shown in Fig- ure 6A, the increase in PA activity induced by SK-Hepl or S180 cell-conditioned medium could be reversed by anti-rbFGF antibody but not by irrelevant IgG. Anti- bFGF IgG also abolished the increase in PAI-1 levels of cells incubated with the conditioned media (data not shown). In contrast, immunoprecipitation with anti- rbFGF antibody and protein A-Sepharose did not abolish the uPA-inducing activity of S180 or SK-Hepl cell- conditioned medium (Fig. 6B). These results showed that the uPA overexpression induced by the tumor cell- conditioned media was mediated by bFGF produced by the endothelial cells.

Characterization of the bFGF- and uPA-inducing activities of SK-Hepl cell-conditioned medium To characterize the factors that mediate bFGF and

uPA up-regulation in endothelial cells, SK-Hepl cell- conditioned medium was subjected to the different treatments described in Materials and Methods. As shown in Figure 7, both the bFGF- and uPA-inducing activities were completely abolished by boiling for 5 min and by trypsin digestion for 30 min. The activities were dialyzable through a 10 kDa dialysis membrane but were retained in a dialysis tube with 3.5 kDa M, cutoff. In control experiments rbFGF (18 kDa) was retained in 10 kDa dialysis tubes. Lowering the pH of the conditioned medium to pH 2.0 for 1 h decreased both activities by approximately 50%. Because several endothelial cell growth factors have affinity for heparin, we also characterized the bFGF- and uPA-inducing activities of SK-Hepl cell-conditioned medium by heparin-Sepharose chromatography. As shown in Figure 8, both activities were retained in the column and could be eluted with 0.6 M NaC1. The fractions eluted with higher NaCl concentrations did not stimulate PA activity in BCE cells both after 4 h and 16 h incubation. Similar results were also obtained by heparin- Sepharose chromatography of larger volumes (up to 12 liters) of SK-Hepl cell-conditioned medium (data not shown). Because acidic FGF and bFGF are eluted from heparin-Sepharose with 1.0 M and 2.5 M NaC1, respec- tively, this finding indicated that these growth factors were not present in the conditioned medium in detectable amounts. Recombinant bFGF, run in the same heparin-Sepharose column, was eluted with 2.0 M NaCl (Fig. BB, dashed line). In addition, rbFGF tested at a saturating concentration (550 pM) did not increase bFGF expression in BCE cells (Fig. 9). These results indicated that the bFGF- and uPA-inducing activities of SK-Hepl cell-conditioned medium are associated with heparin-binding, acid-resistant peptide with a M, significantly lower than 18 kDa and higher than 3.5 kDa.

Because both the bFGF- and the PA-inducing activities of the SK-Hepl cell-conditioned medium were associated with the same polypeptide molecule(s1, we used the PA assay to screen known proteins that have molecular features similar to the bFGF/PA-inducing activity of the SK-Hepl (and possibly the SlSO) cell- conditioned medium. Recombinant human macrophage inflammatory proteins (MIP) 2a and 2b, as well as recombinant mouse MIPla and MIPlb, epidermal growth factor (EGF), insulin-like growth factor 1 (IGFl), and

8 PEVERALI ET AL

Control medium

no addition !! 1 i r relevant IgG 23 + anti-bFGF IgG P I I

Control medium

P 1 . , , , , , , , , , ~ . , , , , , , , . ,

2 4 6 8 1 0 2 4 6 8 1 0

PA a c t i v i t y ( fold i n c r e a s e )

Fig. 6. Effect of anti-rbFGF antibody on the PA activity of BCE cells incubated with tumor cell-conditioned media. Medium conditioned by SK-Hepl or S180 cells was incubated with 10 pgiml of irrelevant IgG or affinity-purified anti-rbFGF IgG in the presence or absence of protein A-Sepharose as described in Materials and Methods. Extracts of BCE cells incubated with SK-Hepl cell-conditioned medium for 4 h or with S180 cell-conditioned medium for 28 h were tested for PA activ-

vascular endothelial cell growth factor (VEGF) were tested for PA-inducing activity on BCE cells a t concentrations ranging from 0.01-500 ng/ml. None of these factors increased PA activity in BCE cells after 4 h of incubation (data not shown). In 28 h incubation experiments the PA activity of BCE cells incubated with 1-100 ngiml of each of the MIPS was decreased by approximately 50% relative to control cells. In addition, antisera to MIPl (a and b) and to MIP2 (a and b) did not neutralize the PA-inducing activity of SK-Hepl cell- conditioned medium (data not shown). Thus, the bFGF/ PA-inducing activity of the tumor cell-conditioned medium did not appear to be associated with any of the cytokines tested.

Induction of in vitro angiogenesis by the bFGF/uPA-inducing factor

To test if the bFGF/uPA-inducing factor possessed angiogenic activity, 500 ml of SK-Hepl cell-conditioned medium was loaded onto a 2 ml heparin- Sepharose column and bound protein was eluted stepwise with 0.6 M, 1.0 M, and 2.5 M NaCl as described in Materials and Methods. The fractions eluted with 1.0 M and 2.5 M NaCl did not contain uPA-inducing activity. The fractions eluted with 0.6 M NaCl that contained uPA-inducing activity were pooled and concentrated in a Centricon tube with 3.5 kDaMr cutoff membrane followed by dialysis with a 10 kDaMr cutoff membrane. The dialysate through the 10 kDaMr cutoff membrane induced a fortyfold increase in the PA activity of BCE cell extracts, whereas the other fractions were ineffec- tive (data not shown). All the fractions were tested for in vitro angiogenesis in three-dimensional fibrin gels. BAE cells grown on fibrin gels for 24 h formed a regular monolayer (Fig. 10A). In the presence of the 10 kDa dialysate single cells began to invade the gel. After 96 h

ity by the '"I-fibrin assay as described in Materials and Methods. A: Conditioned media without addition of protein A-Sepharose. B: Condi- tioned media from which the IgGs were removed with protein A-Sepharose. The data are presented as fold increase relative to the PA activity of cells incubated with control medium. Mean ? s.d. of three experiments is shown.

of incubation the cultures treated with this fraction (Fig. 10C,D) or with SK-Hepl cell-conditioned medium (Fig. 10B) contained numerous cells that formed branched multicellular tube-like structures within the gel beneath the cell layer. In contrast, cultures incubated with control medium or with the other fractions contained occasional, single invasive cells. After 5 days of incubation the cultures were treated with bacterial collagenase as described in Materials and Methods, to remove the layer of non-invasive cells from the gel surface. After this treatment, a considerable number of elongated multicellular structures could be seen in the gel of cultures incubated with the 10 kDa dialysate (Fig. 10F). These structures were virtually absent in the gels of cells incubated with control medium or with the other fractions, and few single cells could be seen (Fig. 10E). This indicated that the cells treated with the active fraction had migrated deeply into the fibrin gel and were inaccessible to collagenase. In contrast, in non-stimulated cultures single invasive cells likely remained on or close to the gel surface and were therefore removed by the enzyme treatment.

Addition of anti-bFGF IgG to cultures treated with the 10 kDa dialysate reduced endothelial cell invasion into the fibrin gel to control levels. Because no bFGF was present in the active fraction from the heparin- Sepharose column, these results showed that the angiogenic effect was mediated by endogenous bFGF overex- pressed by the endothelial cells. Thus, both the bFGFi uPA- and angiogenesis-inducing activities of SK-Hepl cell-conditioned medium are associated with heparin- binding M, 3.5-10 kDa polypeptide molecule(s).

DISCUSSION The data reported show that medium conditioned by

SK-Hepl or S180 cells induces a rapid up-regulation of

9 INDUCTION OF ENDOTHELIAL CELL bFGP AND UROKINASE

bFGF - 2 8 S

- 1 8 s

GAPDH

Q 25001 T

I-

T 2000

c v

a 1500 .- -

0

ul

0

e I

a 1000

.- 2 500

4 0 0

.-

Fig. 7. Characterization of the bFGF- and PA-inducing activities of SK-Hepl cell-conditioned medium. BCE 8A2 cells were incubated for 4 h with control medium or with aliquots of SK-Hepl cell-conditioned medium subjected to the treatments described in Materials and Meth- ods. At the end of the incubation cell extracts were assayed for bFGF mRNA levels by Northern blotting (upper panel) and for PA activity by the Iz5I-fibrin assay (lower panel) as described in Materials and Methods. Control, control medium; cond. med., SK-Hepl cell-conditioned medium, untreated; 100°C (5 min), SK-Hepl cell-conditioned medium boiled for 5 min; pH 2.0 (1 h), SK-Hepl cell-conditioned medium after acidification to pH 2.0 with HCl, incubation a t room temperature for 1 h, and neutralization with NaOH; 3.5 KD dial, SK-Hepl cell-conditioned medium dialyzed in a 3.5 kDa Centricon tube; 3.5 KD ret., SK-Hepl cell-conditioned medium retained in a 3.5 kDa Centricon tube; 10 KD dial, SK-Hepl cell-conditioned medium dialyzed in a 10 kDa Centricon tube; 10 KD ret., SK-Hepl cell-conditioned medium retained in a 10 kDa Centricon tube; Trypsin + ST Inh., SK-Hepl cell-conditioned medium incubated a t 37°C for 30 min with 0.5 mgiml of trypsin and 5 mgiml of soybean trypsin inhibitor; Trypsin, SK-Hepl cell-conditioned medium after incubation a t 37°C for 30 min with 0.5 mgiml of trypsin, followed by addition of 5 mgiml of STI. GAPDH mRNA is shown as a control. The experiment shown in the upper panel was repeated twice, the experiment shown in the lower panel three times with similar results. Mean t- range of variation of duplicate samples from a representative experiment are shown in the lower panel.

bFGF and uPA expression in vascular endothelial cells, as well as formation of capillary-like structures in fibrin gels. In the culture medium of SK-Hepl cells these activities are associated with a peptide that has moderate affinity for heparin and is relatively acid-resistant. Known growth factors with similar but not identical biochemical features, including MIPS, EGF, and IGF1, do not possess this activity. In addition, a number of findings indicate that the bFGF/uPA-inducing activity of SK-Hepl cell-conditioned medium is not associated

with bFGF or TGFpl that are produced by many normal and tumor cells: 1) S180 cell-conditioned medium contained bFGF/uPA-inducing activity, although S180 cells do not appear to express bFGF; 2) no bFGF activity could be recovered from large volumes of SK-Hepl cell-conditioned medium by heparin-Sepharose chromatography; 3) immunoprecipitation with anti-rbFGF antibody and protein A-Sepharose did not abolish the PA-inducing activity of the conditioned media; 4) rbFGF at a saturating concentration did not upregulate bFGF expression in endothelial cells; 5) the bFGF/uPA- inducing activity had an affinity for heparin significantly lower than rbFGF, was dialyzable through 10 kDaMr cutoff membranes by which 18 kDa rbFGF was retained, and was relatively acid-resistant, whereas bFGF is unstable at acidic pH (Basilico and Moscatelli, 1992); 6) the bFGF/uPA-inducing activity up-regulated PA activity in endothelial cells with a time course different from that obtained with rbFGF; 7) TGFpl has a higher M,, is present in most culture supernatants in a latent, inactive form that is activated by acid treatment, and has higher affinity for heparin (McCaffrey et al., 1992). Other heparin-binding cytokines with angiogenic activity, including vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor/ scatter factor (HGF/SF), have M,s considerably higher than the estimated M, of the bFGF/uPA-inducing factor.

Our estimation of the M, of the bFGF/uPA-inducing factor is based on the sizing obtained with different dialysis membranes. The cutoffs of these membranes are not absolute. Therefore, we can estimate that the bFGF/uPA-inducing factor has a M, higher than 3.5 kDa and significantly lower than 18 kDa. It is possible that bFGF fragments derived from proteolytic degradation are dialyzable through 10 kDa membranes and retain biological activity. Basic FGF molecules lacking up to 40 N-terminal or 46 C-terminal amino acid residues have reduced affinity for heparin and a hundred- fold lower biological activity than intact bFGF (Seno et al., 1990). Similarly, bFGF peptides consisting of 10-45 amino acid residues possess biological activity at concentrations at least 1,000-fold higher than the intact molecule (100 Fg/ml to 1 mg/ml) (Baird et al., 1988). Since we could detect biological activity in non-concentrated Sam-ples, it is most unlikely that the tumor cell- conditioned media contained pg/ml concentrations of cleaved bFGF. Furthermore, in cell cultures bFGF is predominantly associated with the glycosaminoglycans of the extracellular matrix and/or the cell surface, and in this form it is protected from proteolytic cleavage (Saksela et al., 1988).

The effect of the tumor cell-conditioned media ap- peared to be more prominent on the 3.7 Kb bFGF mRNA than on the major, 7.0 Kb bFGF transcript of endothelial cells. The role(s) of the two bFGF transcripts and their relative contribution to the expression of the different M, forms of bFGF are not known. There- fore, the biological significance of the differential effect of the tumor cell-conditioned media on the level of the two bFGF transcripts remains to be determined.

The interaction of the tumor-derived factor with endothelial cells results in bFGF overexpression that me- diates an up-regulation of uPA production. Blockade of

10

- 3

- 2

- 1

- 0

PEVERALI ET AL

A c 1 2 3 4 5

bFGF

GAPDH

800

600

400

200

-20s

-18s

0.3

0 co (v

0.2 . 9 0

- c

V 0 Z u

2 4 6 8 1 0 Fract lon No.

Fig, 8. Heparin-Sepharose chromatography of the PA-inducing and bFGF-inducing activities of SK-Hepl cell-conditioned medium. A Basic FGF-inducing activity. SK-Hepl cell-conditioned medium (50 ml) was loaded onto a 1 ml heparin-Sepharose column. Bound protein was eluted stepwise with increasing NaCl concentrations as described in Materials and Methods. BCE 8A2 cells were incubated for 4 h with control medium (C), SK-Hepl cellkondition medium (lane l), SK- Hepl cell-conditioned medium dialyzed vs. column buffer (lane 2), 0.6 M NaCl eluate (lane 3), 1.0 M NaCl eluate (lane 4), or 2.5 M NaCl eluate from the heparin-Sepharose column (lane 5). Basic FGF mRNA was analyzed by Northern blotting as described in Materials and Methods. GAPDH mRNA is shown as a control. This experiment was repeated three times with similar results. B PA-inducing activity.

endothelial cell bFGF results in decreased uPA activity and inhibition of capillary formation in vitro. Thus angiogenesis can be mediated by an up-regulation of endogenous bFGF expression by vascular endothelial cells. With the exception of TGFP1, endothelial cell expression of other cytokines or oncogenes involved in cell proliferation and/or angiogenesis, including TGFa, c-sis, PDGF receptor, K-fgfhst, and c-myc, was not af- fected by the tumor cellkonditioned media. Both bFGF and TGPl are angiogenic in vitro and/or in vivo but have opposing effects on the proteolytic activity of en-

SK-Hepl cellkonditioned medium (50 ml) or 1 ml of control medium containing 1 pg of rbFGF was loaded on a heparin-Sepharose column and eluted with a NaCl gradient as described in Materials and Meth- ods. Undiluted flow-through material or 10 p1 aliquots of the eluate per milliliter of medium were tested for PA-inducing activity on BCE 8A2 cells as described in Materials and Methods. This experiment was repeated five times with similar results. Each point represents mean & range of variation in duplicate samples from a representative experiment (+J, O.D. 280 of SK-Hep 1 cell-conditioned medium; (t), PA-inducing activity of SK-Hep 1 cell-conditioned medium; (--A --J, PA-inducing activity of rbFGF; (. . . .), NACl concentration (moles/ liter).

dothelial cells in vitro (Pepper et al., 1990; Saksela et al., 1987). uPA plays a key role in the invasive pro- cesses that occur during angiogenesis in vitro (Mignatti et al., 1989; Pepper et al., 1992); however, its expression in forming capillaries in vivo appears to be temporally restricted (Bacharach et al., 1992). The increased expression of both bFGF and TGFPl in vascular endothelial cells may provide a mechanism for the regulation of uPA activity during angiogenesis. The uPA produced by endothelial cells stimulated by endogenous bFGF generates high local concentrations of plasmin that ac-


1 2 3

b F G F

- 28s

- 18s

GAPDH

Fig. 9. Effect of pure rbFGF and tumor cell-conditioned medium on bFGF mRNA levels of BCE cells. Northern blotting analysis of bFGF mRNA in BCE 8A2 cells incubated for 4 h with control medium (lane l), SK-Hepl cell-conditioned medium (lane 2), or medium containing 10 ng/ml of human rbFGF (lane 3). RNA extraction, blotting, and hybridization to the 32P-labeled bFGF cDNA probe were carried out as described in Materials and Methods. GAPDH mRNA is shown as a control. This experiment was repeated three times. Similar results were obtained with BAE and GM7373 cells.

tivates TGFPl (Lyons et al., 1990; Flaumenhaft et al., 1992; Sato and Rifkin, 1989; Sato et al., 1990). This growth factor up-regulates PAI-1 and down-regulates uPA expression (Pepper et al., 1990; Saksela et al., 1987). Thus, in endothelial cells bFGF can act both directly as a positive regulator of uPA expression and indirectly, through plasmin generation and TGFPl activation, as a negative modulator of uPA expression and activity.

Whether TGFPl expression is stimulated by the same factor that up-regulates bFGF expression or by a different tumor cell-derived factor remains to be determined. The findings that rbFGF does not stimulate TGFPl synthesis in BCE cells (Flaumenhaft et al., 1992) and that anti-rbFGF antibody does not block TGFPl expression in the same cells (our unpublished results) rule out the hypothesis that TGFPl up-regulation is mediated by endogenous bFGF.

The bFGF-mediated up-regulation of uPA in endothelial cells treated with tumor cell-conditioned medium raises interesting points as to the mechanism of action of endogenous bFGF in these cells. The kinetics of uPA induction by SK-Hepl or S180 cell-conditioned medium were not superimposable to that obtained by addition of exogenous rbFGF to endothelial cells. These features of uPA overexpression indicate that endogenous bFGF may act through a mechanism(s) different from that (those) of exogenous bFGF. The difference between the effect of SK-Rep1 cell-conditioned medium and that of S180 cell-conditioned medium on endothelial cell PA activity can be explained by several hypotheses: 1) the two tumor cell lines secrete different bFGF/uPA-stimulating factors or different amounts of the same bFGF/uPA-inducing factor( s); 2) they contain different factors or different concentrations of a factor(s) that interferes with the bFGF/uPA-inducing activity; or 3) the two conditioned media contain a differ-

ent factorb) or different concentrations of a factor that regulates additional components of the PA system (e.g., PAI-1 or the uPA receptor [uPAR]). On the basis of our results, we cannot discriminate between these hypotheses. Preliminary data (not shown) indicate that the bFGF/uPA-inducing activity of S180 cell-conditioned medium has biochemical properties similar to that of SK-Hepl cell-conditioned medium. Both conditioned media up-regulate PAI-1 mRNA levels in endothelial cells (Fig. 4). Although no uPA/PAI-1 complexes were detected by fibrin zymography (Fig. 5 ) , PAI-1 production might be upregulated in the endothelial cells with kinetics different from uPA and be modulated by the two conditioned media in a different manner. In addition, we found that both SK-Hepl and S180 cell-conditioned media induce increased levels of uPAR in BAE and GM7373 cells. However, the effect of SK-Hepl cell- conditioned medium on uPAR expression appears to be comparable to that of S180 cell-conditioned medium (our unpublished results).

The inhibition of endothelial cell PA activity by anti- bFGF antibody is consistent with previous findings (Sasada et al., 1988; Sato and Rifkin, 1988) and prompts consideration of the mechanismb) by which bFGF is externalized by endothelial cells. Basic FGF lacks a secretory signal peptide (Abraham et al., 1986); however, significant amounts of the growth factor are associated with the ECM of endothelial cells (Bashkin et al., 1989; Flaumenhaft et al., 1989; Rogelj et al., 1989; Vlodavski et al., 1987) and with the basal lamina of blood vessels (Cordon et al., 1990; DiMario et al., 1989; Folkman et al., 1988). Although cell death or sublethal cell injury may represent a mechanism of bFGF release in cell cultures (McNeil et al., 1989; Sch- weigerer et al., 1987; Witte et al., 1989), bFGF released by single, isolated cells modulates their migration in an autocrine manner (Mignatti et al., 1991b). It has been proposed that the growth factor is externalized via a mechanism of exocytosis independent of the classical endoplasmic reticulum-Golgi pathway (Mignatti and Rifkin, 1991; Mignatti et al., 1992). Thus, our data showing inhibition of PA activity by anti-bFGF antibody provide further evidence that bFGF can modulate endothelial cell functions in an autocrine manner.

During tumor angiogenesis, capillary formation pro- ceeds through alternate cycles of activation and depres- sion of different cell functions. Repeated cycles lead to formation of a capillary network (Ausprunk et al., 1975; Ausprunk and Folkman, 1977; Folkman, 1986). The coordinated cascade of angiogenic factor expression and proteinase-proteinase inhibitor-mediated growth factor activation reactions in endothelial cells treated with tumor cell-conditioned medium may provide the biochemical basis for the alternate cycles of ECM degradation-invasion, and cell quiescence-ECM deposition. We showed that this cascade can be triggered in endothelial cells by a peptide factorb) secreted by tumor cells that up-regulates bFGF expression in vascular endothelial cells.

Thus, angiogenesis can be mediated by an autocrine mechanism resulting from the overexpression of endogenous bFGF by vascular endothelial cells, rather than or in addition to a paracrine effect of bFGF derived from other cells or ECM. This conclusion is consistent with

12 PEVERALI ET AL.

Fig. 10. In vitro angiogenesis in three-dimensional fibrin gels. BAE cells were grown on three-dimensional fibrin gels in control medium (A,E), SK-Hepl celkonditioned medium (B), or in medium supplemented with 100 Fliml of protein purified from SK-Hepl cell-conditioned medium as described in Results (C,D,F). The cells were photographed in phase contrast after 4 days of incubation a t 37°C (A-D).

After 5 days of incubation, the cell layer was removed from the gel surface as described in Materials and Methods, and the gels were photographed in phase contrast (E,F). In photographs B-F the plane of focus is beneath the cell surface. A: X125, B-D: ~ 2 4 0 . E,F: x40. Representative fields are shown. This experiment was repeated four times; similar results were also obtained with BCE cells.

13 INDUCTION OF ENDOTHELIAL CELL bFGF AND UROKINASE

the finding of elevated levels of bFGF in the capillary endothelial cells of some tumors (Schulze-Osthoff et al., 1990). The purification and characterization of this tumor-derived angiogenesis factor will offer insights into blood vessel formation and may provide tools to control tumor growth and metastasis.

ACKNOWLEDGMENTS The second and third author of this paper contributed

equally to the work. This work was supported by grants from the Italian Association for Cancer Research (AIRC) and the Italian Ministry of University, and Sci- entific and Technological Research (MURST 60%) to P.M., from the Italian National Research Council (CNR) (Progetto: Applicazioni Cliniche della Ricerca Oncologica) and AIRC to G.D.V., and from the National Institutes of Health and the American Cancer Society to D.B.R. We are indebted to Drs. R. Dono, T. Fotsis, D. Moscatelli, and L. Philipson for critically reading the manuscript, to Prof. L. De Carli for helpful discussions, and to M. Negri, E. Toufexis, F. Tredici, and M. Vas- sallo for their skilful technical assistance. Part of the work was carried on during P.M.’s and R.M.’s tenure at NYU Medical Center. F.P. and P.C. were recipients of fellowships from AIRC.

LITERATURE CITED Abraham, J.A., Mergia, A,, Whang, J.L., Tumolo, A,, Friedman, J.,

Hjerrild, K., Gospodarowicz, D., and Fiddes, J . (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science, 233545-548.

Auffray, C., and Rougeon, F. (1980) Purification of mouse immuno- globulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem., 107t303-314.

Ausprunk, D.H., and Folkman, J . (1977) Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res., 14t53-65.

Ausprunk, D.H., Knighton, D.R., and Folkman, J . (1975) Vasculariza- tion of normal and neoplastic tissues grafted into the chick chorioal- lantois. Am. J . Pathol., 79t597-618.

Bacharach, E., Itin, A., and Keshet, E. (1992) In vivo patterns of expression of urokinase and its inhibitor PAL1 suggest a concerted role in regulating physiological angiogenesis. Proc. Natl. Acad. Sci. U.S.A., 89:1068&10690.

Baird, A., Schubert, D., Ling, N., and Guillemin, R. (1988) Receptor- and heparin-binding domains of basic fibroblast growth factor. Proc. Natl. Acad. Sci. U.S.A., 85t232P-2328.

Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, C.M., Folkman, J., and Vlodavski, I. (1989) Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry, 28t1737-1743.

Basilico, C., and Moscatelli, D. (1992) The FGF family of growth factors and oncogenes. Adv. Cancer Res., 59~115-165.

Cordon, C.C., Vlodavsky, I., Haimovitz, F.A., Hicklin, D., and Fuks, Z. (1990) Expression of basic fibroblast growth factor in normal human tissues. Lab. Invest., 63:832-840.

DiMario, J . , Buffinger, N., Yamada, S., and Strohman, R.C. (1989) Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle. Science, 244t688-690.

Flaumenhaft, R., Moscatelli, D., Saksela, O., and Rifkin, D.B. (1989) Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator and DNA synthesis. J. Cell. Physiol., 140: 75-81.

Flaumenhaft R., Moscatelli, D., and Rifkin, D.B. (1990) Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor. J. Cell Biol., 111:1651-1659.

Flaumenhaft, R., Abe, M., Mignatti, P., and Rifkin, D.B. (1992) Basic fibroblast growth factor-induced activation of latent transforming growth factor p in endothelial cells: Regulation of plasminogen activator activity. J . Cell Biol., 118:901-909.

Folkman, J. (1986) How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes Memorial Award LEcture. Cancer Res., 46:467-473.

Folkman, J . (1990) What is the evidence that tumors are angiogenesis dependent? J . Natl. Cancer Inst., 82:4-6 [editorial].

Folkman, J. , and Klagsbrun, M. (1987) Angiogenic factors. Science, 235:442-447.

Folkman, J. , Haudenschild, C.C., and Zetter, B. (1979) Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. U.S.A., 76521 7-522 1.

Folkman, J. , Klagsbrun, M., Sasse, J., Wadzinski, M., Ingber, D., and Vlodavski, I. (1988) A heparin-binding angiogenic protein-basic fibroblast growth factor-is stored within basement membrane. Am. J . Pathol., 130t393-400.

Folkman, J., Watson, K., Ingber, D., and Hanahan, D. (1989) Induc- tion of angiogenesis during the transition from hyperplasia to neo- plasia. Nature, 339.58-61.

Gospodarowicz, D., Massoglia, S., Cheng, J., Lui, G.-M., andBohlen, P. (1985) Isolation of bovine pituitary fibroblast growth factor purified by fast protein liquid chromatography (FPLC). Partial chemical and biological characterization. J . Cell. Physiol., 122t323-328.

Granelli-Piperno, A,, and Reich, E. (1978) A study of proteases and protease inhibitor complexes in biological fluids. J. Exp. Med., 148: 223-234.

Gross, J., Moscatelli, D., Jaffe, E.A., and Rifkin, D.B. (1982) Plasmino- gen activator and collagenase production by cultured capillary endothelial cells. J . Cell Biol., 95:97&981.

Lyons, R.M., Gentry, L.E., Purchio, A.F., and Moses, H.L. (1990) Mechanism of activation of latent recombinant transforming growth factor p1 by plasmin. J. Cell Biol., 110t1361-1367.

McCaffrey, T.A., Falcone, D.J., and Du, B. (1992) Transform- ing growth factor-beta 1 is a heparin-binding protein: Identifica- tion of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-beta 1. J . Cell. Physiol., 152r430- 440.

McNeil, P.L., Muthukrishnan, L., Warder, E,. and DAmore, P. (1989) Growth factors are released by mechanically wounded endothelial cells. J . Cell Biol., 109t811-822.

Mignatti, P., and Rifkin, D.B. (1991) Release ofbasic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: A trivial phenomenon or a novel secretion mechanism? J . Cell. Bio- chem., 47t201-207.

Mignatti, P., and Rifkin, D.B. (1993) Biology and biochemistry of proteinases in tumor invasion. Physiol. Rev., 73:161-195.

Mignatti, P., Robbins, E., and Rifkin, D.B. (1986) Tumor invasion through the human amniotic membrane: Requirement for a proteinase cascade. Cell, 47t487-498.

Mignatti, P., Tsuboi, R., Robbins, E., and Rifkin, D.B. (1989) In vitro angiogenesis on the human amniotic membrane: Requirement for basic fibroblast growth factor-induced proteinases. J . Cell. Biol., 108:671-682.

Mignatti, P., Mazzieri, R,. and Rifkin, D.B. (1991a) Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J . Cell Biol., 113t1193-1201.

Mignatti, P., Morimoto, T., and Rifkin, D.B. (1991b) Basic fibroblast growth factor released by single, isolated cells stimulates their migration in an autocrine manner. Proc. Natl. Acad. Sci. U.S.A., 88: 11007-11011.

Mignatti, P., Morimoto, T., and Rifkin, D.B. (1992) Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J . Cell. Physiol., 151t81-93.

Montesano, R., Vassalli, J.D., Baird, A., Guillemin, R., and Orci, L. (1986) Basic fibroblast growth factor induces angiogenesis in vitro. Proc. Natl. Acad. Sci. U.S.A., 83:7297-7301.

Montesano, R., Pepper, M.S., Vassalli, J.-D., and Orci, L. (1987) Phor- bol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors. J . Cell. Physiol., 132t509- 516.

Moscatelli, D., Presta, M., Joseph-Silverstein, J., and Rifkin, D.B. (1986a) Both normal and tumor cells produce basic fibroblast growth factor. J . Cell. Physiol., 129:273-276.

Moscatelli, D., Presta, M., and Rifkin, D.B. (1986b) Purification of a factor from human placenta that stimulates endothelial cell protease production, DNA synthesis, and migration. Proc. Natl. Acad. Sci. U.S.A., 83t2091-2095.

Pepper, M.S., Belin, D., Montesano, R., Orci, L., and Vassalli, J.D. (1990) Transforming growth factor-beta 1 modulates basic fibro-

PEVERALI ET AL 14

blast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J. Cell Biol., 111:743-755.

Pepper, M.S., Sappino, A.P., Montesano, R., Orci, L., and Vassalli, J.D. (1992) Plasminogen activator inhibitor-1 is induced in migrating endothelial cells. J. Cell. Physiol., 153t129-139.

Rogelj, S., Klagsbrun, M., Atzmon, R., Kurokawa, M., Haimovitz, A,, Fuks, Z., and Vlodavski, I. (1989) Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells. J . Cell Biol., 109323-831.

Saksela, O., and Rifkin, D.B. (1990) Release ofbasic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J. Cell Biol., 110: 767-775.

Saksela, O., Moscatelli, D., and Rifkin, D.B. (1987) The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells. J. Cell Biol., 105:957-963.

Saksela, O., Moscatelli, D., Sommer, A,, and Rifkin, D.B. (1988) En- dothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J . Cell Biol., 107:743-751.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Extraction, purification, and analysis of messenger RNA from eukaryotic cells. In: Molecular Cloning. A Laboratory Manual. J . Sambrook, E.F. Fritsch and T. Maniatis, eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 1, pp. 7.1-7.87.

Sasada, R., Kurokawa, T., Iwane, M., and Igarashi, K. (1988) Trans- formation of mouse BALBic 3T3 cells with human basic fibroblast growth factor cDNA. Mol. Cell. Biol., 8588-594.

Sato, Y., and Rifkin, D.B. (1988) Autocrine activities of basic fibroblast growth factor: Regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J. Cell Biol., 107: 1199-1205.

Sato, Y., and Rifkin, D.B. (1989) Inhibition of endothelial cell movement by pericytes and smooth muscle cells: Activation of a latent transforming growth factor-pl-like molecule by plasmin during coculture. J. Cell Biol., 109t309-315.

Sato, Y., Tsuboi, R., Lyons, R.M., Moses, H.L., and Rifkin, D.B. (1990) Characterization of the activation of latent TGF-P by co-cultures of endothelial cells and pericytes or smooth muscle cells: A self-regulating system. J. Cell Biol., lllt757-763.

Sato, Y., Shimada, T., and Takaki, R. (1991) Autocrinological role of

basic fibroblast growth factor on tube formation of vascular endothelial cells in vitro. Biochem. Biophys. Res. Commun., 180r1098- 1102.

Schulze-Osthoff, K., Risau, W., Vollmer, E., and Sorg, C. (1990) In situ detection of basic fibroblast growth factor by highly specific antibodies. Am. J. Pathol., 137t85-92.

Schwartz, S. (1978) Selection and characterization of bovine aortic endothelial cells. In Vitro, 14t966980.

Schweigerer, L., Neufeld, G., Friedman, J,. Abraham, J.A., Fiddes, J.C., and Gospodarowicz, D. (1987) Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature, 325257-259.

Seno, M., Sasada, R., Kurokawa, T., and Igarashi, K. (1990) Carboxyl- terminal structure of basic fibroblast growth factor significantly contributes to its affinity for heparin. Eur. J . Biochem., 188:239- 245.

Shing, Y., Folkman, J., Haudenschild, C., Lund, D., Crum, R., and Klagsbrun, M. (1985) Angiogenesis is stimulated by a tumor-derived cell growth factor. J. Cell. Biochem. 29:275-287.

Sternfield, M.D., Hendrikson, J.E., Keeble, W.W., Rosenbaum, J.T., Robertson, J.E., Pittelkow, M.R., and Shipley, G.D. (1988) Differen- tial expression of mRNA coding for heparin-binding growth factor type 2 in human cells. J . Cell. Physiol., 136:297-304.

Vlodavski, I., Folkman, J. , Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J . , and Klagsbrun, M. (1987) Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. U.S.A., 84: 2292-2296.

Vlodavski, I., Korner, G., Ishai, M.R., Bashkin, P., Bar, S.R., and Fuks, Z. (1990) Extracellular matrix-resident growth factors and enzymes: Possible involvement in tumor metastasis and angiogenesis. Cancer Metastasis Rev., 9:203-226.

Vlodavski, I., Fuks, Z., Ishai, M.R., Bashkin, P., Levi, E., Korner, G., Bar, S.R., and Klagsbrun, M. (1991) Extracellular matrix-resident basic fibroblast growth factor: Implication for the control of angiogenesis. J. Cell. Biochem., 45:167-176.

Weidner, N., Semple, J.P., Welch, W.R., and Folkman, J. (1991) Tu- mor angiogenesis and metastasis4orrelation in invasive breast carcinoma. N. Engl. J. Med., 324:l-8.

Witte, L., Fuks, Z., Haimovitz, F.A., Vlodavski, I., Goodman, D.S., and Eldor, A. (1989) Effects of irradiation on the release of growth factors from cultured bovine, porcine, and human endothelial cells. Cancer Res., 49:5066-5072.

Tumor cells secrete an Angiogenic factor that stimulates basic fibroblast growth factor and...

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