FGF-2 stimulates migration of Kaposi's sarcoma-like vascular cells by HGF-dependent relocalization of the urokinase receptor

^a Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy
^b Department of Morphology, University of Geneva Medical Center, Geneva, Switzerland
^c Department of Biomedical Sciences and Technologies, University of Milano, Milano, Italy

	ABSTRACT

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The spindle-shaped cell line TTB was recently isolated from highly vascularized skin lesions of BKV/HIV-1 tat transgenic mice and shown to possess an autocrine loop for hepatocyte growth factor (HGF). We show that fibroblast growth factor-2 (FGF-2) stimulates TTB cell migration and promotes polarization of uPAR at the leading edge of migrating cells. FGF-stimulated TTB cells presented the typical migratory phenotype, with a triangular cell shape and concomitant breakdown of actin stress fibers and smooth muscle-specific actin isoform. FGF-2-stimulated migration was blocked by antibodies against urokinase-type plasminogen activator (uPA) or uPA receptor (uPAR) and by neutralizing anti-HGF antibodies. The latter also inhibited uPAR relocalization at the cell surface of FGF-2-treated TTB cells. This points to a crosstalk between FGF-2 and HGF that might mediate TTB cell migration by modulating the localization of cell surface uPAR.—Cavallaro, U., Wu, Z., Di Palo, A., Montesano, R., Pepper, M. S., Maier, J. A. M., Soria, M. R. FGF-2 stimulates migration of Kaposi's sarcoma-like vascular cells by HGF-dependent relocalization of the urokinase receptor. FASEB J. 12, 1027–1034 (1998)

Key Words: TTB cell migration • hepatocyte growth factor • FGF • uPAR relocalization

	INTRODUCTION

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FIBROBLAST GROWTH FACTORS (FGFs)² are involved in the pathogenesis of various diseases characterized mainly by exaggerated neovascularization (1), including Kaposi's sarcoma (KS) (2). The latter is a disease frequently associated with acquired immunodeficiency syndrome (AIDS), characterized by the presence of skin and mucosal patches containing many blood vessels and various cell types (3). HIV-1 Tat protein has been thought to play a role in the pathogenesis of AIDS-associated KS (2), and the development of KS-like lesions in tat transgenic mice apparently confirms this hypothesis (4, 5). It has been proposed that FGF-2 synergizes with HIV-1 Tat in initiating and maintaining KS lesions (6). However, since human herpesvirus 8 is frequently detectable in KS samples from both HIV-positive and -negative patients (3), some yet uncharacterized viral factor (or factors) other than HIV-1 Tat may participate in recruiting endothelial and other cell types in the lesions of KS.

Spindle-shaped cells, which could to be the proliferating component of KS (3), have been detected in skin lesions of BKV/tat transgenic mice, and the spindle KS-like TTB cell line was isolated from these lesions (7). We previously characterized these cells as having several characteristics in common with human spindle cells isolated from KS lesions (8). Upon subcloning TTB cells, we selected one clone for further investigation. This TTB clone, as well as the parental cell line, synthesizes and secretes hepatocyte growth factor (HGF) and expresses the HGF receptor, the product of the oncogene c-met (9), at the cell surface (10, 11). Since FGF-2 is mitogenic for human KS cells (12), we investigated the effects of FGF-2 on TTB cells and show that FGF-2 causes cell migration and relocalization of the urokinase receptor (uPAR), both of which are mediated by HGF.

	METHODS

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Materials
Recombinant FGF-2 was either the kind gift of P. Sarmientos and L. Garofano (Pharmacia-Upjohn, Milano, Italy) or was purchased from Boehringer-Mannheim (Mannheim, Germany). Anti-phosphotyrosine monoclonal antibodies PY20 and 4G10 were from Transduction Laboratories (Lexington, Ky.) and Upstate Biotechnology (Lake Placid, N.Y.), respectively. Rabbit anti-mouse uPAR and anti-mouse uPA were the kind gifts of S. Rosenberg (Chiron Corp., Emeryville, Calif.) and G. Høyer-Hansen (Copenhagen, Denmark), respectively. Rabbit antiserum against HGF ß-chain was kindly provided by A. Galvani (Pharmacia-Upjohn). This antiserum also recognizes unprocessed, single-chain HGF (13). Neutralizing antibodies against HGF were goat immunoglobulin G (IgG) and a mouse monoclonal antibody (clone 24612.111) of the IgG1 subtype (R&D Systems, Minneapolis, Minn.). Nonimmune goat and rabbit IgG were purified by protein G-Sepharose (Pharmacia, Uppsala, Sweden) chromatography from whole sera (Sigma, St. Louis, Mo.). The monoclonal IgG1 B4E11 against chromogranin A was kindly provided by A. Corti (Milano). The monoclonal IgG1 1B8 has been described previously (14).

Cells
TTB cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, Md.) supplemented with 10% (v/v) fetal calf serum (Sigma). We subcloned TTB cells by isolating single colonies from parental TTB cells in order to obtain a homogeneous cell population. One clone coexpressing endothelial, smooth muscle, and antigen-presenting cell markers, as previously reported for TTB cells (8), was selected for further investigation.

Immunofluorescence staining
Cells were seeded onto glass coverslips and treated with recombinant FGF-2, as described above. At the end of the treatment, cells were washed and fixed in phosphate-buffered saline, pH 7.6, containing 3% (w/v) paraformaldehyde and 2% (w/v) sucrose. Cells were then permeabilized as previously described (15) before incubation with primary antibodies. Tetramethylrhodamine isothiocyanate (TRITC) -labeled secondary antibodies were from Dakopatts (Glostrup, Denmark). Cells were routinely counterstained with fluorescein isothiocyanate (FITC) -labeled phalloidin (Sigma) to visualize F-actin.

Cell migration assay
The assay was performed by a modification of a previously described technique (16). Briefly, TTB cells were seeded onto glass coverslips and, upon confluence, single wounds were made across cell monolayers to create a cell-free path. Thereafter, cells were incubated for 24 h in DMEM containing 1% fetal calf serum (FCS) in either the presence or absence of 15 ng/ml FGF-2. At the end of treatment, cells were fixed, permeabilized, and stained with FITC-conjugated phalloidin (see above).

Collagen gel invasion assay
TTB cells were seeded on the surface of collagen gels, prepared as described (17), and stimulated with FGF-2 for 7 days. Five randomly selected fields measuring 0.5 x 0.7 mm were photographed in each dish at a focal level 30 µm beneath the surface monolayer; cell invasion was assessed by counting the number of cells per microscopic field.

Immunoprecipitation and Western blotting analyses
Control or FGF-2-treated subconfluent cells were washed and lysed in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na₃VO₄, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors from Complete kit (Boehringer-Mannheim). Cell lysates were centrifuged to remove cell debris. For immunoblotting analyses, total cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose at 150 mA for 16 h. Blots were then probed with specific antibodies. In immunoprecipitation experiments, cell lysates were precleared with nonimmune serum and protein G-Sepharose, and then incubated with anti-phosphotyrosine monoclonal antibodies PY20 and 4G10. The immune complexes were bound to protein G-Sepharose and eluted in Laemmli buffer at 95°C for 5 min. Samples were electrophoresed and blotted as described above. Finally, blots were probed with anti-mouse c-Met antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.), followed by peroxidase-conjugated secondary antibodies (Pierce, Rockford, Ill.). The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins.

Scatter assay
TTB cells were incubated for 48 h in DMEM containing 1% FCS in either the presence or absence of 15 ng/ml recombinant FGF-2. At the end of the treatment, conditioned media were harvested and centrifuged. Scatter assays on Madin-Darby canine kidney (MDCK) epithelial cells were used to assess HGF biological activity (18), performed as described previously (10). MDCK cells were incubated in DMEM, 1% FCS, and treated for 24 h with conditioned media from untreated or FGF-2-treated TTB cells. Results were always evaluated by a blinded observer. FGF-2 added to MDCK cell cultures had no effect on scattering (not shown).

	RESULTS

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FGF-2 stimulates TTB cell migration concomitant with cytoskeletal alterations
Spindle-shaped TTB cells were weakly growth-stimulated by FGF-2 (not shown), but FGF-2 caused marked phenotypic changes in these cells. Unstimulated TTB cells show a well-organized cytoskeleton, with abundant stress fibers organized into bundles; they synthesize high amounts of -smooth muscle actin, which colocalizes with F-actin in the stress fibers (8). However, when treated with FGF-2, TTB cells showed a triangular cell shape and a marked decrease in stress fibers characteristic of the motile and migratory phenotype (19) ( Fig. 1B see also Fig. 3), and migrated faster than untreated control cells after wounding of confluent monolayers ( Fig. 1A, B). Immunoreactivity of -smooth muscle actin decreased concomitant with the disassembly of stress fibers in FGF-2-treated cells ( Fig. 1D), but the overall amount of -smooth muscle actin ( Fig. 1E) and total actin (not shown) was not affected by FGF-2 treatment in total cell lysate immunoblotting experiments. Recombinant FGF-1 induced the same morphological changes in TTB cells as FGF-2, whereas HGF, vascular endothelial growth factor (VEGF), and placenta growth factor (PlGF) had no effect on cell morphology (not shown). Unlike VEGF and PlGF, however, HGF stimulated TTB cell migration in the wounding assay, although to a lesser extent than FGF-2 (not shown).

View larger version (140K): [in this window] [in a new window]   Figure 1. Effects of FGF-2 on TTB migration. Confluent monolayers were wounded as described in Materials and Methods and incubated in DMEM containing 1% FCS, in either the absence (A) or presence (B–D) of 15 ng/ml FGF-2 for 24 h. Cells were stained with FITC-labeled phalloidin (A–C) and with a monoclonal antibody against -smooth muscle actin followed by TRITC-labeled anti-mouse secondary antibody (D). E) 50 µg of total protein from lysates of cells treated with FGF-2 for the indicated periods of time were electrophoresed under reducing conditions and immunoblotted with anti--smooth muscle actin antibody. A, B) 480x; C, D) 2000x.

View larger version (108K): [in this window] [in a new window]   Figure 3. Effects of FGF-2 on uPAR distribution in TTB cells. Cells seeded on coverslips were incubated either in the absence (A, B) or presence (C, D) of 15 ng/ml FGF-2 for 24 h. For HGF neutralization, cells were pretreated with goat anti-HGF IgG (40 µg/ml) for 2 h prior to addition of FGF-2 (E, F). Cells were then costained with FITC-labeled phalloidin (A, C) and anti-uPAR antibody, followed by TRITC-labeled anti-rabbit secondary antibody (B, D). Focal adhesion sites in nonmigrating cells are indicated (small arrows), as well as the leading edge (large open arrows) and lagging strand (large solid arrows) of migrating cells. 2000x.

To confirm the results of the wounding assays in a 3-dimensional system and to quantify the effects of FGF-2 on TTB cell migration, we performed assays of collagen gel invasion. As shown in Fig. 2, FGF-2 stimulated TTB invasion of gels in a dose-dependent fashion, with a peak at 10 ng/ml FGF-2.

View larger version (62K): [in this window] [in a new window]   Figure 2. Effects of FGF-2 on gel invasion by TTB cells. Cells seeded on the surface of collagen gels were stimulated with the indicated concentrations of FGF-2 for 7 days. Thereafter, invading cells were counted as described in Materials and Methods. Data correspond to the means ± SEM from three separate experiments (i.e., a total of 15 microscope fields per experimental condition). *P < 0.001 vs. untreated cells.

The uPA/uPAR system is involved in FGF-2-stimulated cell migration
FGF-2 is known to up-regulate the expression of uPA in vascular endothelial cells (20–22). To verify whether urokinase-mediated plasminogen activation played a role in the faster migration and phenotypic changes induced in TTB cells by FGF-2, wounding assays were performed in the presence of antibodies against either uPA or uPAR. Anti-uPA or anti-uPAR antibodies efficiently decreased the migration of FGF-2-treated cells, whereas nonimmune rabbit IgG had no effect ( Table 1). We then investigated whether FGF-2 affected the levels of uPA, uPAR, and/or plasminogen activator inhibitor 1 (PAI-1) in TTB cells. In immunoblotting experiments, intracellular accumulation of uPA was increased in FGF-2-stimulated TTB and in human umbilical vein endothelial (HUVE) cells, with a maximal effect at 24 h after addition of FGF-2 (not shown). Finally, by analogy to previous observations on the expression of PAI-1 in microvascular endothelial cells (22), FGF-2 induced an early (8 h) and transient increase of PAI-1 in TTB cells, whereas maximal effects on HUVE cells were observed at 24 h (not shown).

uPAR is redistributed in migrating TTB cells
As described in vascular endothelial and smooth muscle cells (21, 23, 24), FGF-2 up-regulated the expression of uPAR in TTB cells (not shown). In addition, FGF-2 dramatically affected the cellular localization of uPAR. In untreated cells, uPAR was mostly localized at cell-to-substratum adhesion sites, both on the ventral face and at the periphery of cells ( Fig. 3B). Upon FGF-2 treatment, uPAR appeared enriched at the leading edge and at the lagging strand of migrating cells ( Fig. 3D), indicating that FGF-2-induced migration of TTB cells involved relocalization of uPAR. This effect was already detectable after 8 h of FGF-2 treatment (not shown), concomitant with the appearance of cells with a triangular shape, and reached a maximum after 24 h, when most cells showed the motile phenotype. FGF-2 specifically affected the subcellular localization of uPAR. Indeed, distribution at the cell surface of LDL receptor-related protein (also known as ₂-macroglobulin receptor) did not change significantly upon cell stimulation with FGF-2 (not shown).

uPA was detected mostly with an intracellular and perinuclear localization, but could also be detected occasionally at the invading front of migrating cells (not shown). The distribution of PAI-1, localized primarily in the extracellular matrix, was not affected by FGF-2 (not shown).

HGF mediates migration of FGF-2-treated TTB cells
We reasoned that the presence of an autocrine circuit for HGF might play a role in the stimulating effects of FGF-2 on TTB cell migration. Therefore, we verified whether we could affect FGF-2-induced migration of TTB by neutralizing secreted HGF. As shown in Table 1, migration of FGF-2-treated cells was blocked either by anti-HGF polyclonal IgG or by neutralizing monoclonal antibodies, whereas nonimmune goat IgG and two irrelevant monoclonal antibodies had no effect. Disassembly of actin stress fiber and shape changes caused by FGF-2 treatment were not inhibited by anti-HGF antibodies ( Fig. 3E).

FGF-2 enhances HGF accumulation and secretion by TTB cells
Immunoblotting experiments confirmed the accumulation of cell-associated HGF by FGF-2-treated TTB cells previously described in other cell types (13) ( Fig. 4A). HGF is synthesized as an inactive, single-chain polypeptide (pro-HGF) of about 90 kDa. Upon proteolytic activation, HGF becomes a disulfide-linked heterodimer composed by a 60 kDa -chain and a 34 kDa ß-chain (9). Both pro-HGF and activated HGF were increased in FGF-2-treated TTB cells, as observed with an HGF ß-chain-specific antiserum ( Fig. 4A). In addition, FGF-2 induced the expression of c-Met in TTB ( Fig. 4B, lower panel) concomitant with an increase in the amount of activated c-Met ( Fig. 4B, upper panel), assessed by its tyrosine phosphorylation (9).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of FGF-2 on accumulation of HGF and c-Met and on the activation of c-Met in TTB cells. Cells were treated with 15 ng/ml FGF-2 for the times indicated. A) 100 µg aliquots of total protein from lysates of TTB cells treated as above were electrophoresed under reducing conditions and immunoblotted with anti-HGF serum. B, upper panel) Cell lysates (600 µg aliquots of total proteins) were subjected to immunoprecipitation with a mixture of anti-phosphotyrosine monoclonal antibodies PY20 and 4G10 (1 µg each). SDS-PAGE was performed under nonreducing conditions, followed by immunoblotting with anti-mouse c-Met. B, lower panel) To evaluate total amounts of c-Met, 50 µg aliquots of total protein from lysates of TTB cells treated as described above were electrophoresed under reducing conditions and immunoblotted with anti-mouse c-Met.

These observations suggested that FGF-2 might stimulate secretion of biologically active HGF by TTB cells. To verify this hypothesis, conditioned media of untreated and FGF-2-treated cells were tested for scattering activity toward MDCK cells (18). Conditioned media from TTB induced MDCK cell scattering in a dose-dependent fashion (not shown), as described for TTB cells (10). Scattering was more prominent after incubation with conditioned media from FGF-2-treated TTB cells than from untreated cells. When anti-HGF antibodies were added to TTB cells 2 h before treatment with FGF-2, scattering activity was abolished, whereas nonimmune IgG had no effect ( Table 2). Finally, preincubating conditioned medium from FGF-2-treated TTB cells with neutralizing anti-HGF antibodies specifically inhibited MDCK scattering in a dose-dependent fashion (not shown). These results indicate that FGF-2 stimulates TTB cells to secrete biologically active HGF. Immunoblotting experiments confirmed the increase of HGF levels in conditioned media from FGF-2-treated cells (not shown).

HGF mediates uPAR relocalization in FGF-2-stimulated TTB cells
To clarify the role of HGF in the migration of FGF-2-treated TTB cells, we assessed whether HGF neutralization might prevent up-regulation of uPA upon cell stimulation with FGF-2. We found that anti-HGF neutralizing antibodies had no effect on uPA levels, as judged by immunoblotting analysis (not shown). We then investigated the effect of neutralizing HGF on uPAR relocalization at the cell surface of TTB cells treated with FGF-2. The localization of uPAR at the leading edge was inhibited in most TTB cells either by anti-HGF polyclonal IgG ( Fig. 4F) or by neutralizing monoclonal antibodies, whereas nonimmune goat IgG and two irrelevant monoclonal antibodies had no effect (data not shown). Only uPAR at the leading edge of TTB cells was affected by anti-HGF; uPAR at the lagging strand could still be visualized ( Fig. 3F, large solid arrows) when all uPAR at the leading edge had disappeared.

	DISCUSSION

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We have demonstrated that TTB cells respond to exogenously added FGF-2 by assuming a migratory phenotype as well as by faster migration in wounding assays, and that the latter effect is blocked by antibodies against uPA or uPAR. An increase in uPA accumulation, concomitant with cell migration, is observed in FGF-2-treated cells, as reported for other cell types (22, 25). However, FGF-2 did not increase the enzymatic activity of secreted uPA (not shown). Lack of FGF-2-mediated activation of uPA has been reported on other cell types, such as smooth muscle cells, which migrate in response to FGF-2 (26). In addition, uPA-mediated migration of endothelial cells upon FGF-2 stimulation is independent of uPA proteolytic activity (27).

FGF-2 affected the cellular localization of surface-bound uPA, since migrating TTB cells polarized uPAR at their leading edge. Localization of uPAR at the invasive front of moving cells, as well as of growing tumor masses (28), has been reported in several experimental models (29–31). Thus, FGF-2 might stimulate TTB cell migration through a pathway common to other cell types.

The inhibitory effects on migration by anti-HGF neutralizing antibodies indicated that secreted HGF is essential for TTB cells to migrate. Like other heparin binding growth factors, secreted HGF can be sequestered by heparan sulfate proteoglycans (HPSGs) of the extracellular matrix (9), from which it is released upon matrix degradation during cell migration. FGF-2 induced an increase in the level of HGF, both in cell lysates and in conditioned media from TTB. This effect of FGF-2 might depend on increased synthesis and secretion of HGF by TTB cells and/or on the release of HGF from HSPGs associated with the cell surface or the extracellular matrix. Therefore, uPA-mediated local proteolysis after FGF-2 treatment of TTB cells might modulate the release of HGF from HPSGs in the microenvironment of migrating cells. Indeed, HGF induces uPA and uPAR expression in epithelial cells (32), and extracellular uPA has been reported to activate HGF, with the two molecules forming a complex at the cell surface (33). Thus, neutralization of uPA might inhibit TTB cell migration due not only to reduction of plasmin-mediated matrix degradation possibly affecting release of HGF, but also to a decrease in activation of HGF by uPA.

HGF neutralization prevented the relocalization of uPAR to the leading edge of migrating cells upon FGF-2 stimulation. This observation suggests that HGF mediates FGF-2-induced TTB cell migration by modulating the surface distribution of uPAR rather than by up-regulating uPA and/or its receptor. In this fashion, HGF would control the spatial distribution of cell surface-bound proteolysis without affecting overall proteolytic activity. In addition, the interaction between uPA and HGF at the cell surface (33) might imply that uPAR redistribution upon stimulation of TTB cells with FGF-2 serves to localize uPA-bound HGF at restricted pericellular areas. Therefore, HGF might mediate cell migration through uPAR relocalization, with uPAR contributing to focalizing HGF activity of specific compartments at the cell surface.

The role of HGF in FGF-2-induced migration or proliferation of endothelial and smooth muscle cells was not anticipated based on previous observations. Both HGF and FGF-2 stimulate endothelial cell growth in an additive manner, whereas only FGF-2 induces smooth muscle cell proliferation (34, 35). Thus, the crosstalk that we observed between FGF-2 and HGF might reflect the peculiar coexpression of endothelial, smooth muscle, and antigen-presenting cell markers in TTB (8).

Similar to what has been observed in other cell types (13, 36, 37), FGF-2 stimulated accumulation and secretion of HGF by TTB cells, as well as accumulation and activation of c-Met. We previously reported a similar autocrine HGF/c-Met up-regulation by interleukin 1 (IL-1) on TTB cells that did not result in cell growth stimulation (10) or cell migration (U. Cavallaro, unpublished observation). This raises the interesting possibility that FGF-2 and IL-1 might up-regulate HGF/c-Met in TTB cells through some shared intracellular signaling pathway. This pathway would then diverge downstream, resulting in migration only when originated with signaling by FGF.

Coexpression of HGF and c-Met has been reported in various tumor cell types (38, 39), but a crosstalk between FGF-2 and HGF was not reported. TTB cells are not tumorigenic (7), and this might account for differences in the response to FGF-2 by TTB involving HGF-mediated uPAR relocalization. An HGF autocrine loop has recently been described in myoblasts (40). These cells might represent a suitable model to extend our observations on FGF-2-stimulated TTB cells to other nontransformed cell types and to clarify the role of the autocrine HGF circuit in uPAR relocalization.

	ACKNOWLEDGMENTS

 
We are grateful to G. Distefano for subcloning TTB cells and to P. C. Marchisio and F. Blasi for helpful discussions and suggestions. We thank P. Sarmientos, L. Garofano, S. Rosenberg, G. Høyer-Hansen, A. Galvani, and A. Corti for providing reagents. This research was supported by Progetto AIDS 1996–1997, Ministero della Sanità, Rome, Consiglio Nazionale delle Ricerche-Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica (CNR-PF ACRO) to M.R.S. and J.A.M.M., by Associazione Italiana per la Ricerca sul Cancro (AIRC) to J.A.M.M., and a grant from the Swiss National Science Foundation to R.M. and M.S.P.; U.C. is supported by a fellowship from Istituto Superiore di Sanità, Rome.

	FOOTNOTES

 
¹ Correspondence: Dibit, San Raffaele Scientific Institute, Via Olgettina 58, I-20132 Milano, Italy. E-mail: cavallu{at}dibit.hsr.it

² Abbreviations: AIDS, acquired immunodeficiency syndrome; FGF, fibroblast growth factor; KS, Kaposi's sarcoma; HGF, hepatocyte growth factor; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor; Ig, immunoglobulin; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MDCK, Madin-Darby canine kidney; VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PAI-1, plasminogen activator inhibitor 1; HUVE, human umbilical vein endothelial; HPSG, heparan sulfate proteoglycan.

Received for publication November 24, 1997. Accepted for publication March 18, 1998.

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