Noncompetitive, Chemokine-mediated Inhibition of Basic Fibroblast Growth Factor-induced Endothelial Cell Proliferation*

Marco PrestaDagger §, Mirella BelleriDagger , Annunciata Vecchi, Joseph Hesselgesserpar , Alberto MantovaniDagger , and Richard Horukpar

From the Dagger  Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia 25123, Italy, the  Istituto Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy, and the par  Department of Immunology, Berlex Biosciences, Richmond, California 94804

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The proinflammatory and chemoattractant chemokine interleukin-8 (IL-8) inhibits cell proliferation induced by basic fibroblast growth factor (bFGF) in mouse endothelial cells isolated from subcutaneous sponge implant (sponge-induced mouse endothelial cells) and in bovine aortic endothelial GM 7373 cells. The mechanism of action of IL-8 was investigated in GM 7373 cells. IL-8 did not prevent the binding of bFGF to its tyrosine kinase FGF receptors (FGFRs) nor to cell surface heparan sulfate proteoglycans (HSPGs). A transient interaction of IL-8 with the cell before the addition of the growth factor was sufficient to prevent bFGF activity. The inhibitory activity of IL-8 was abolished by protein kinase C (PKC) inhibitors and was mimicked by the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Accordingly, both IL-8 and 12-O-tetradecanoylphorbol-13-acetate caused a ~60% decrease of the binding capacity of GM 7373 cells due to the down-regulation of FGFRs. Several C-X-C and C-C chemokines exerted an inhibitory action on bFGF activity similar to IL-8. Soluble heparin, 6-O-desulfated heparin, N-desulfated heparin, and heparan sulfate but not 2-O-desulfated heparin, chondroitin-4-sulfate, hyaluronic acid, and K5 polysaccharide abrogated IL-8 inhibitory activity consistently with the presence of low affinity, high capacity HSPG-like chemokine-binding sites on GM 7373 cells. Finally, neovascularization induced by bFGF in murine subcutaneous sponge implants was reduced significantly by IL-8. In conclusion, IL-8 inhibits the mitogenic activity exerted by bFGF on cultured endothelial cells by a PKC-dependent, noncompetitive mechanism of action that causes FGFR down-regulation. This activity is shared by several chemokines and requires endothelial cell surface HSPGs. The endothelial cell line utilized in the present study may help to elucidate the complex interplay among chemokines, HSPGs, growth factors, and receptors in endothelial cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The process of angiogenesis, in which new capillary blood vessels are formed, is strictly regulated in the adult, and the proliferation of endothelial cells is low compared with many other cell types. Notable exceptions to this tight regulation of angiogenesis are found in the female reproductive system and during wound healing. The consequences of uncontrolled endothelial cell proliferation manifest themselves in tumor neovascularization and in angioproliferative diseases. The control of angiogenesis is achieved by a balance between the levels of angiogenic and angiostatic factors that modulate neovascularization both in physiological and pathological conditions.

Several molecules including basic fibroblast growth factor (bFGF)1 have been demonstrated to be positive mediators of angiogenesis in vitro and in vivo (1). bFGF belongs to the FGF family that includes at least nine different gene products that share a high affinity for heparin and a partial amino acid sequence homology (2). bFGF exerts angiogenic activity in vivo and induces cell proliferation, chemotaxis, and protease production in cultured endothelial cells (3) by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) and low affinity proteoglycans (HSPGs) containing heparan sulfate (HS) polysaccharides (4, 5). The interaction of bFGF with FGFRs is modulated by HSPGs leading to the formation of bFGF·HSPG·FGFR ternary complexes (5). When the binding of bFGF to low affinity HSPGs is prevented by treating the cells with heparinase or chlorate, the binding of bFGF to FGFRs is also reduced together with its ability to stimulate cell proliferation (6). In addition HSPGs may induce FGF(s) oligomerization, which facilitates FGFR dimerization and signal transduction (7).

Chemokines belong to a superfamily of low molecular weight chemotactic proteins that are active on different leukocyte populations. They are divided into three structural subclasses depending on whether the first two of the four invariant cysteine residues are adjacent (C-C chemokines), separated by an intervening residue (C-X-C chemokines) or whether the first cysteine is missing (C chemokines) (8-10). Moreover, C-X-C chemokines are subdivided further depending on the presence or the absence of the sequence Glu-Leu-Arg (the ELR motif) (8). Chemokine receptors are members of the G protein-coupled seven-transmembrane family, and they bind either C-C or C-X-C chemokines (11). An exception to this ligand specificity is the promiscuous Duffy antigen/erythrocyte chemokine receptor (DARC), which can bind members of both the C-X-C and C-C class (12). So far no physiological function for DARC has been established. Besides a high affinity receptor interaction, chemokines bind sulfated glycosaminoglycans (GAGs) including heparin and HS (13) and thus can interact with HSPGs of the cell membrane and extracellular matrix. HSPGs on endothelial cells have been shown to present some chemokines to leukocytes in the multistep process of recruitment (14).

Recent work indicates that various chemokines may affect endothelial cell function. In particular, interleukin-8 (IL-8) and other C-X-C chemokines that contain the ELR motif (ELR+ C-X-C chemokines) have been reported to induce endothelial cell chemotaxis and/or proliferation in vitro and angiogenesis in vivo (15-17). In contrast, platelet factor-4 (PF-4) and other C-X-C chemokines lacking the ELR motif (ELR- C-X-C chemokines) exert an angiostatic activity in vivo and inhibit bFGF-induced proliferation and migration in cultured endothelial cells (17, 18). However, the molecular basis underlying the pro- and anti-angiogenic activity of chemokines has not been clearly established, and some contradictory findings have been reported (see "Discussion"). The high degree of heterogeneity observed for endothelial cells of different tissue origins isolated from different animal species and the significant differences between large vessel and microvascular endothelium (3, 4) may explain, at least in part, these discrepancies. Also, the use of human umbilical vein endothelial cells (HUVECs) obtained from different individuals and cultured under different conditions probably underlies the conflicting and confusing evidence available on the interaction of chemokines with endothelial cells (19). Unclear results have been obtained also concerning the expression and function of chemokine-binding sites in endothelial cells. DARC has been detected in endothelial cells of post-capillary venules and splenic sinusoids (20), whereas the presence of high affinity binding sites for 125I-IL-8, together with IL-8 receptor type II mRNA expression, has been reported in human vascular endothelial cells (21) but not confirmed by others (22). The capacity of interferon-gamma -inducible protein-10 (IP-10) to share with PF-4 a specific HSPG-binding site in SV-40-transformed murine endothelial cells has been also demonstrated (23).

In this study we demonstrate that IL-8 inhibits bFGF-induced cell proliferation in two out of six endothelial cell types tested. The bFGF antagonist activity exerted by IL-8 on fetal bovine aortic endothelial GM 7373 cells was due to a protein kinase C (PKC)-dependent, noncompetitive mechanism of action whose activation causes FGFR down-regulation. Various ELR+ C-X-C, ELR- C-X-C, and C-C chemokines share with IL-8 the capacity to inhibit bFGF activity in GM 7373 cells. A promiscuous HSPG-like chemokine-binding site was identified on these cells by binding assays with radiolabeled IL-8, growth-related oncogene-alpha /melanoma growth stimulating activity (GROalpha /MGSA), and RANTES and by competition experiments with free GAGs. Finally, neovascularization induced by bFGF in murine subcutaneous sponge implants was reduced significantly by IL-8, in keeping with its capacity to inhibit the mitogenic activity exerted in vitro by bFGF on mouse endothelial cells isolated from these sponges.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Human recombinant bFGF was expressed and purified from transformed Escherichia coli cells by heparin-Sepharose affinity chromatography (24). Phorbol esters, epidermal growth factor, dibutyryl cAMP, and 5'-methyl-thioadenosine were from Sigma. Suramin was from Bayer AG (Leverkuse, Germany). H-7 and H-8 were from Siekagaku America (Ijamsville, MD). Bisindolylmaleimide GF 109203X was a gift of Dr. M. Pizzi (Institute of Pharmacology, University of Brescia, Brescia, Italy). Chemokines were from PeproTech Inc. (Rocky Hill, NC). Heparin was obtained from a commercial batch preparation of unfractionated sodium heparin from beef mucosa (1131/900 from Laboratori Derivati Organici S.p.A., Milan, Italy). Type I heparan sulfate (HS) was from Opocrin (Corlo, Italy). K5 polysaccharide was a gift of Dr. G. Zoppetti (Glycosaminoglycan Consultants, Milan, Italy). Chondroitin-4-sulfate and hyaluronic acid were a gift of Dr. M. Del Rosso (University of Florence, Florence, Italy). 2-O-desulfated, 6-O-desulfated, totally O-desulfated, and N-desulfated heparins were provided by Dr. B. Casu (Ronzoni Institute, Milan, Italy). The characteristics of the GAGs utilized in the present study have been described elsewhere (25).

Cell Cultures

Fetal bovine aortic endothelial GM 7373 cells, corresponding to the described BFA-1c 1BPT clone (26), were obtained from the National Institute of General Medical Sciences, Human Genetic Mutual Cell Repository (Camden, NJ) and grown in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum (FCS), vitamins, and essential and nonessential amino acids. Adult bovine aortic endothelial cells (BAECs) were grown in MEM containing 10% FCS and were utilized at passages 10 and 11. HUVECs were grown in M199 containing 100 µg/ml heparin, 150 µg/ml bovine brain extract, and 20% FCS and were utilized at passages 3 and 4. Balb/c mouse brain microvascular endothelial 10027 cells (MBECs) were provided by Dr. R. Auerbach (University of Wisconsin) and were grown in Dulbecco's modification of MEM added with 10% FCS (27). 1G11 cells, an endothelial cell line cloned from the lung of C57BL/6NCrLBR mice (28) and microvascular endothelial cells isolated from subcutaneous sponges implanted in mice of the same strain (SIECs) (28) were grown in Dulbecco's MEM supplemented with 1 mM glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, and 20% FCS in the presence of 10 ng/ml bFGF and 10 µg/ml heparin on gelatin-coated dishes.

Cell Proliferation Assays

GM 7373 Cells-- The short term cell proliferation assay was performed as described (29, 30). Briefly, 70,000 cells/cm2 were seeded in 96-well dishes. After overnight incubation, the cells were incubated for 24 h in fresh medium containing 0.4% FCS and 10 ng/ml bFGF in the presence of increasing concentrations of different chemokines. At the end of the incubation, the cells were trypsinized and counted in a Burker chamber. During this time period, control cultures incubated with no addition or with 10 ng/ml bFGF undergo 0.1-0.2 and 0.7-0.8 cell population doublings, respectively. Cells grown in 10% FCS undergo 1.0 cell population doublings (30). For long term proliferation studies, GM 7373 cells were seeded in 24-well plates at 10,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Medium was changed every 72 h, and cells were trypsinized and counted after 6 days of treatment.

BAECs-- Cells were seeded in 35-mm dishes at 2,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 6 days, cells were trypsinized and counted.

HUVECs-- Cells were seeded in 96-well plates at 6,250 cells/cm2. After 16 h, cells were incubated in fresh medium with 5% calf serum in the presence of 10 ng/ml bFGF and/or 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 6 days, cells were trypsinized and counted.

MBECs-- Cells were seeded in 24-well plates at 25,000 cells/cm2. After 16 h, cells were incubated in fresh medium with 10% FCS in the presence of 10 ng/ml bFGF and of 100 ng/ml IL-8. Then medium with or without the growth factor and chemokine was changed every other day. After 3 days, cells were trypsinized and counted (27).

1G11 Cells-- They were seeded in gelatin-coated 48-well plates at 8,000 cells/cm2. After 16 h, cells were incubated in fresh medium without heparin containing 20% FCS in the presence of 10 ng/ml bFGF and 100 ng/ml IL-8. After 2 days, cells were trypsinized and counted.

SIECs-- Cells were seeded in gelatin-coated 48-well plates at 10,000 cells/cm2. After 16 h, cells were incubated in fresh medium without heparin containing 20% FCS in the presence of 10 ng/ml bFGF and increasing concentrations of IL-8. After 3 days, cells were trypsinized and counted.

125I-bFGF Binding Assay

Human recombinant bFGF was labeled with 125I (37 GBq/ml; Amersham International) using Iodogen (Pierce) as described (31) at a specific radioactivity equal to 600 cpm/fmol. GM 7373 cells were seeded in 24-well dishes at the density of 75,000 cells/cm2. After 24 h, cells were washed three times with cold PBS and incubated for 2 h at 4 °C in binding medium (serum-free medium containing 0.15% gelatin, 20 mM Hepes buffer, pH 7.5) with 10 ng/ml 125I-bFGF in the presence of increasing concentrations of the chemokine under test. After a PBS wash, cells were then washed twice with M NaCl in 20 mM Hepes buffer (pH 7.5) to remove 125I-bFGF bound to low affinity HSPGs and twice with 2 M NaCl in 20 mM sodium acetate (pH 4.0) to remove 125I-bFGF bound to high affinity FGFRs (32). Nonspecific binding was measured in the presence of a 100-fold molar excess of unlabeled FGF-2 and subtracted from all the values.

Down-regulation of FGFRs

GM 7373 cells were plated in 24-well dishes at a density of 70,000 cells/cm2. After 24 h, cells were incubated at 37 °C for 6 h with 100 ng/ml IL-8 or TPA. At the end of the incubation, the cells were washed twice with ice-cold 2.0 M NaCl in 20 mM sodium acetate, pH 4.0, and three times with ice-cold PBS and then incubated at 4 °C in binding medium containing increasing concentrations of 125I-bFGF. After 2 h, the amount of 125I-bFGF bound to high and low affinity sites was measured as described above. Binding data were analyzed by the Scatchard plot procedure (33).

Western Blot Analysis of Extracellular Signal-regulated Kinase-2 (ERK-2)

GM 7373 cells were grown at subconfluence in 60-mm dishes in Dulbecco's MEM containing 10% FCS. Then cells were treated for 20 min with fresh medium with or without 10 ng/ml bFGF in the absence or in the presence of 100 ng/ml IL-8. Western blot analysis of the cell extracts was performed exactly as described (34) using anti-ERK-2 antibodies (kindly provided by Dr. Y. Nagamine, Friedrich Miescher Institute, Basel, Switzerland). Phosphorylation of ERK-2 was evidenced as a mobility shift on the gel (34).

Chemokine Binding Assays

GM 7373 cells (1 × 106 cells/ml) were incubated in suspension with 125I-labeled ligands (0.5 nM) and varying concentrations of unlabeled ligands at 4 °C for 1 h. The incubation was terminated by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously (35). Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve fit with the computer program IGOR (Wavemetrics) to determine the affinity (KD), the number of sites, and nonspecific binding.

Subcutaneous Sponge Assay

Fragments (about 1 cm3) of sponge (Spongostan Dental, Ferrosan, Denmark) hydrated for 2 h at 4 °C in saline alone or saline containing 100 ng of bFGF, 1 µg of IL-8, or bFGF plus IL-8 (200 µl/sponge) were inserted subcutaneously in the flank of anesthetized Crl:CD1(ICR)BR mice (Charles River, Milan, Italy). All sponges also contained 200 µg of heparin to stabilize the bFGF molecule in vivo (36). After 7 days sponges were removed, and neovascularization was quantified by measuring their hemoglobin content as described (36).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

IL-8 Inhibits the Mitogenic Activity of bFGF-- A high degree of heterogeneity has been observed in endothelial cells originating from different tissues, vessels of different caliber, or different animal species (37). The effect of IL-8 on endothelial cell proliferation was therefore evaluated on cells of different origin. Primary HUVEC, BAEC, 1G11, and SIEC cultures and immortalized MBEC and fetal bovine aortic endothelial GM 7373 cell lines were incubated with the chemokine in the absence or in the presence of 10 ng/ml bFGF. As shown in Fig. 1, 100 ng/ml IL-8 did not affect the proliferation of HUVECs, BAECs, MBECs, and 1G11 cells when administered either in the absence or in the presence of bFGF. In contrast, IL-8 abolished the mitogenic activity exerted by bFGF in SIECs and GM 7373 cells with an ID50 of 30 ng/ml both in short term and in long term cell proliferation assays (Fig. 1). Heat inactivation abolished the capacity of IL-8 to inhibit the mitogenic activity of bFGF (data not shown), indicating that an appropriate three-dimensional conformation is required for the chemokine to elicit its inhibitory activity. No morphologic signs of apoptosis, including cell rounding and detachment, were detected even after 6 days of IL-8 treatment in all cell types investigated. These observations prompted us to more carefully characterize the mechanism of action of IL-8. To this purpose, the short term (24 h) cell proliferation assay performed on the fetal bovine aortic endothelial GM 7373 cell line was used routinely.


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Fig. 1.   Effect of IL-8 on endothelial cell proliferation. HUVECs, BAECs, MBECs, and 1G11 cells were incubated with no addition (black bar), 100 ng/ml IL-8 (open bar), 10 ng/ml bFGF (dashed bar), or bFGF plus IL-8 (dotted bar) as described under "Experimental Procedures." At the end of incubation, cells were trypsinized and counted. Each value is the mean ± S.E. of three determinations. In parallel experiments SIECs and endothelial GM 7373 cells were incubated with 10 ng/ml bFGF in the presence of increasing concentrations of IL-8. GM 7373 cell mitogenic response was assessed both in a short term (open circle ) and in a long term (bullet ) cell proliferation assay. Data are expressed as percentages of the stimulation of cell proliferation exerted by bFGF alone when compared with untreated cultures. The results are representative of three independent experiments, and the variation was less than 15%.

The inhibitory effect exerted by IL-8 on bFGF-induced GM 7373 cell proliferation was dose-dependent (Fig. 1) and was not reverted by increasing concentrations of bFGF (Fig. 2A), suggesting that the mechanism of action of IL-8 is not competitive. Accordingly, IL-8 did not prevent the binding of 125I-bFGF to high affinity tyrosine kinase FGFRs even when administered at doses 10 times higher than those required to fully inhibit the mitogenic activity of bFGF (Fig. 2B). Also, ERK-2 phosphorylation, an early step in the signal transduction pathway activated by FGFR after ligand interaction (32), was retained in GM 7373 cells incubated with bFGF in the presence of IL-8 (Fig. 2B, inset). Taken together, the data indicate that IL-8 does not directly compete for the binding of bFGF to its tyrosine kinase receptors in GM 7373 cells. Also, IL-8 did not prevent the binding of 125I-bFGF to HSPGs in GM 7373 cell cultures (Fig. 2B), in keeping with its lower affinity for sulfated GAGs when compared with bFGF (IL-8 and bFGF elute from heparin-Sepharose columns at 0.5 and 2.0 M NaCl, respectively (38, 39)).


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Fig. 2.   The inhibitory activity of IL-8 on bFGF-induced GM 7373 cell proliferation is not competitive. A, GM 7373 cells were incubated with no IL-8 (black-square) or with 20 (open circle ), 100 (triangle ), and 300 (black-triangle) ng/ml IL-8 in the presence of increasing concentrations of bFGF. After 24 h, cells were trypsinized and counted. Data are expressed as percentages of the stimulation of cell proliferation exerted by 10 ng/ml bFGF alone when compared with untreated cultures. The results are representative of three independent experiments. B, GM 7373 cells were incubated for 2 h at 4 °C with 10 ng/ml 125I-bFGF in the presence of increasing concentrations of IL-8. Then radioactivity associated with low affinity HSPGs (black-triangle) and high affinity FGFRs (bullet ) was evaluated. Nonspecific binding values evaluated in the presence of 1 µg/ml unlabeled bFGF were subtracted from the total binding to yield specific binding values. Each point is the average of three wells, and variability among wells was less than 10%. Inset, whole cell extracts from GM 7373 cell cultures treated with 10 ng/ml bFGF (lane a), 100 ng/ml IL-8 (lane b), bFGF plus IL-8 (lane c), or vehicle (lane d) were analyzed by Western blot using antibodies against ERK-2. Activation of ERK-2 is evidenced as a mobility shift on the gel (arrowhead).

To evaluate the timing required by IL-8 to exert its inhibitory activity, GM 7373 cells were exposed to the chemokine at different times before or after the beginning of bFGF treatment. As shown in Fig. 3A, IL-8 fully inhibits the mitogenic activity of bFGF when administered to cell cultures before or together with the growth factor. In contrast, IL-8 exerted only a very limited inhibitory effect when administered 3-6 h after bFGF, thus indicating that the chemokine mainly affects an early event required by bFGF to exert its mitogenic activity in GM 7373 cells.


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Fig. 3.   Effect of IL-8 pretreatment on bFGF mitogenic activity. In A, GM 7373 cells were incubated with 100 ng/ml IL-8 at the indicated times before (+) or after (-) addition of 10 ng/ml bFGF to the culture medium (time 0). 24 h after bFGF addition, cells were trypsinized and counted. In B, GM 7373 cells were incubated with 100 ng/ml IL-8 at the indicated times before addition of 10 ng/ml bFGF to the culture medium (time 0). Immediately before bFGF addition, cell cultures were were washed for 2 min at room temperature with 300 µg/ml suramin to remove both free and cell surface-bound IL-8. Then cells were incubated with bFGF in the absence of any further addition of the chemokine. After 24 h, cells were trypsinized and counted. Data are expressed as percentages of the stimulation of cell proliferation exerted by bFGF alone when compared with untreated cultures. The results are the averages of triplicate wells, and the variation among wells was less than 20%. Similar results were obtained in two independent experiments.

To evaluate the shortest time interval required by IL-8 to elicit its inhibitory effect, GM 7373 cell cultures were incubated with the chemokine at 37 °C for different periods of time ranging from 5 to 360 min before the beginning of bFGF treatment. At the end of the preincubation period, taking advantage of the capacity of suramin to displace heparin-binding proteins from HSPGs and high affinity receptors (30, 40, 41), cells were washed for 2 min at room temperature with 300 µg/ml suramin to remove both free and cell surface-bound IL-8. Cells were then further incubated for 24 h with bFGF in the absence of any further addition of the chemokine. As shown in Fig. 3B, shortening of the preincubation period results in a progressive decrease of the inhibitory capacity of IL-8, maximal inhibition of bFGF activity being observed after 3-6 h of preincubation with the chemokine. Control experiments demonstrated that the suramin wash did not affect bFGF activity (data not shown).

Role of Protein Kinase C in the Inhibitory Activity of IL-8 on GM 7373 Cells-- The above data indicate that a 3-h interaction of IL-8 with GM 7373 cells is sufficient to prevent bFGF-induced cell proliferation and that the presence of the chemokine is not required during the incubation with the growth factor, further supporting the hypothesis that the mechanism of inhibition of IL-8 is not competitive. We took advantage of these characteristics to attempt to define the mechanism of action of IL-8 by incubating GM 7373 cells with putative inhibitors of IL-8 activity during a 3-h period of preincubation at 37 °C with the chemokine. Then cell cultures were washed extensively to remove the chemokine and its inhibitor before the 24-h incubation with bFGF. Under these experimental conditions, the inhibitors tested were anticipated to affect only IL-8 activity without affecting bFGF activity.

PKC participates in chemokine-dependent signal transduction (11) and plays a role in the IL-8-dependent inhibition of IL-4-induced growth of human B cells (42). On this basis, we evaluated the effect of different protein kinase inhibitors on IL-8-mediated inhibition of GM 7373 cell proliferation. As shown in Fig. 4A, the PKC inhibitors H-7 (43) and bisindolylmaleimide GF 109203X (GF 109) (44) abolished the inhibitory effect of IL-8 preincubation on bFGF mitogenic activity. In contrast, the PKA inhibitor N-[2-methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) (43) and the tyrosine kinase inhibitors genistein (45, 46) and 5'-methylthioadenosine (47) were ineffective. It must be pointed out that none of the inhibitors were able to affect the mitogenic activity of bFGF when administered to GM 7373 in the absence of IL-8 under the same experimental conditions. Conversely, the PKC activator phorbol ester TPA was able to mimic the activity of IL-8. Indeed, as observed for IL-8, 3 h of preincubation with 100 ng/ml TPA fully inhibited the mitogenic activity of bFGF in GM 7373 cells, whereas the inactive phorbol esters alpha -phorbol 12,13-didecanoate, phorbol 12,13-diacetate, and phorbol were ineffective (Fig. 4B). Again, the inhibitory activity of TPA was suppressed by the PKC inhibitor GF 109 (Fig. 4B). Taken together, the data indicate that PKC activation may play a role in mediating the inhibitory activity exerted by IL-8 on bFGF-dependent cell proliferation in endothelial GM 7373 cells.


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Fig. 4.   Role of PKC in mediating the inhibitory activity of IL-8. In A, GM 7373 cells were incubated for 3 h at 37 °C with protein kinase inhibitors H-7 (50 µM), GF 109 (2 µM), H-8 (50 µM), genistein (25 µg/ml) or 5'-methyl-thioadenosine (MTA, 1 mM) in the absence (hatched bar) or in the presence (black bar) of 100 ng/ml IL-8. In B, GM 7373 cells were incubated for 3 h at 37 °C with 100 ng/ml IL-8 or with 100 ng/ml phorbol esters TPA (in the absence or in the presence of 2 µM GF 109), phorbol 12,13-diacetate (PDA), alpha -phorbol 12,13-didecanoate (PDD), or phorbol (Pho). In both experiments, cells were then washed extensively and incubated for further 24 h with 10 ng/ml bFGF. At the end of the incubation, cells were trypsinized and counted. Data are expressed as percentages of the stimulation of cell proliferation exerted by bFGF alone when compared with untreated cultures. The results are the averages of triplicate wells, and the variation among wells was less than 15%. Similar results were obtained in three independent experiments.

IL-8 Pretreatment Decreases the Binding Capacity of FGFR-- Transmodulation of tyrosine kinase receptors by PKC activation has been reported (48, 49). To assess whether the inhibitory activity of IL-8 on bFGF mitogenic activity could be explained by a PKC-dependent decrease in FGFR binding capacity, GM 7373 cells were preincubated for 6 h at 37 °C with 100 ng/ml IL-8 or TPA. After extensive washing of the cell monolayers, cells were further incubated for 2 h at 4 °C with 30 ng/ml 125I-bFGF. As anticipated, both molecules caused a significant decrease in the amount of 125I-bFGF bound to high affinity FGFRs (45 and 57% inhibition for IL-8 and TPA, respectively) without affecting the ability of 125I-bFGF to bind to low affinity HSPGs (Fig. 5A). Scatchard plot analysis of the 125I-bFGF binding data, obtained when the experiment was repeated in the presence of increasing concentrations of the radiolabeled ligand, indicated that IL-8 pretreatment caused a significant decrease (~60%) in the number of FGFRs present on the surfaces of GM 7373 cells without affecting their affinity for 125I-bFGF. IL-8 pretreatment did not cause any significant modifications in the number of 125I-bFGF-binding cell surface HSPGs and in their affinity for the growth factor (Table I).


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Fig. 5.   IL-8 decreases the binding capacity of FGFRs. A, GM 7373 cells were incubated for 6 h at 37 °C with 100 ng/ml IL-8, 100 ng/ml TPA, or no addition. Then cells were washed with ice-cold 2.0 M NaCl in 20 mM sodium acetate, pH 4.0, and with ice-cold PBS and then incubated at 4 °C in binding medium containing 30 ng/ml 125I-bFGF. After 2 h, the amount of 125I-bFGF specifically bound to FGFRs (black bar) and HSPGs (dashed bar) was measured. The data are expressed as percentages of the radioactivity specifically bound to GM 7373 cells in the absence of any preincubation. The results are the means ± S.E. of four independent experiments in triplicate. B, GM 7373 cells were incubated with 10 ng/ml bFGF, 10 µM dibutyryl cAMP (db-cAMP), or 10% FCS in the absence or in the presence of 100 ng/ml IL-8. After 24 h, cells were trypsinized and counted. Data are expressed as percentages of cell proliferation exerted by the mitogen alone. The results are the means ± S.E. of two independent experiments in triplicate.

                              
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Table I
Effect of IL-8 on 125I-bFGF binding to GM 7373 cells
GM 7373 cells plated in 24-well dishes at 70,000 cells/cm2 were incubated for 6 h at 37 °C with vehicle or with 100 ng/ml of IL-8. At the end of the incubation, cells were washed twice with ice-cold 2.0 M NaCl in 20 mM sodium acetate, pH 4.0, and three times with ice-cold PBS and then incubated at 4 °C in binding medium containing increasing concentrations of 125I-bFGF. After 2 h, the amount of 125I-bFGF bound to high (FGFRs) and low (HSPGs) affinity sites was measured. Binding data were analyzed by the Scatchard plot procedure.

To confirm the hypothesis that the PKC-dependent mechanism of action of IL-8 depends, at least in part, on a decrease in tyrosine kinase receptor binding capacity, the effect exerted by the chemokine on the mitogenic activity of bFGF was compared with that exerted on the mitogenic activity of two tyrosine kinase receptor-independent mitogenic stimuli represented by dibutyryl cAMP and FCS. As shown in Fig. 5B, when administered to GM 7373 cells together with the mitogen, 100 ng/ml IL-8 caused an approximately 90% decrease in the mitogenic activity of 10 ng/ml bFGF without exerting a significant inhibitory activity on cell proliferation induced by 10 µM dibutyryl cAMP or by 10% FCS.

Both C-X-C and C-C Chemokines Inhibit bFGF Mitogenic Activity in GM 7373 Cells-- IL-8 belongs to the ELR+ subclass of the C-X-C chemokines. To assess whether the capacity to inhibit the mitogenic activity exerted by bFGF in GM 7373 was shared by other chemokines, the ELR+ C-X-C chemokines GROgamma , GROalpha /MGSA, the GROalpha mutant E6A (50), the ELR- C-X-C chemokine PF-4, and the C-C chemokines RANTES, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha (MIP1alpha ) were compared with IL-8 in the short term proliferation assay (Fig. 6). Like IL-8, most of the chemokines tested inhibit bFGF activity with an ID50 equal to 20-40 ng/ml (congruent 3 nM), the exceptions being MIP1alpha and monocyte chemoattractant protein-1 that half-maximally inhibit cell proliferation at 100 and 1000 ng/ml, respectively. It is interesting to note that despite their similar ID50 values, IL-8, GROalpha /MGSA, GROgamma , PF-4, and RANTES cause 80-95% inhibition of GM 7373 cell proliferation at 1000 ng/ml, whereas cells treated with the same dose of MIP1alpha or with the GROalpha E6A mutant still retain 40% of their proliferative response.


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Fig. 6.   Effect of different chemokines on bFGF-induced proliferation in GM 7373 cells. Cells were incubated for 24 h with 10 ng/ml bFGF in the presence of increasing concentrations of the C-X-C chemokines IL-8 (bullet ), PF-4 (triangle ), GROgamma (open circle ), GROalpha /MGSA (black-square), or GROalpha E6A mutant (black-triangle) (left) or of the C-C chemokines RANTES (bullet ), monocyte chemoattractant protein-1alpha (open circle ), or MIP1alpha (black-triangle) (right). After 24 h, cells were trypsinized and counted. Data are expressed as percentages of the stimulation of cell proliferation exerted by bFGF alone when compared with untreated cultures. The results are the averages of triplicate wells, and the variation among wells was less than 10%. For each chemokine, similar results were obtained in three or four independent experiments.

Identification of a Promiscuous Chemokine-binding Site in GM 7373 Cells-- The capacity of both C-X-C and C-C chemokines to exert a similar inhibitory effect on endothelial GM 7373 cells pointed to the possibility that these cells express DARC, as observed for post-capillary venule and splenic sinusoid endothelial cells (20). However, Northern blot analysis failed to detect DARC mRNA in GM 7373 cells (data not shown). Also, GM 7373 cells do not express the C-X-C receptors IL-8-A and IL-8RB (11) as assessed by Northern blot analysis using the corresponding cDNA probes.2 These data prompted us to study the IL-8 binding properties of this endothelial cell line (Fig. 7A). Scatchard plot analysis indicates that 125I-IL-8 binding to GM 7373 cells is both saturable and linear, consistent with a single class of binding sites (900,000 sites/cell) having a KD of 132 ± 16 nM, significantly higher than the KD of the C-X-C receptors IL-8RA and IL-8RB (3-5 nM, Ref. 11) Analysis performed with the C-X-C chemokine 125I-GROalpha /MGSA confirmed the presence of a single class of binding sites on the GM 7373 cell surface (KD equal to 150 ± 13 nM) ranging from 600,000 to 1,000,000 sites/cell. Scatchard analysis with 125I-RANTES yielded similar results (data not shown). The ability of unlabeled chemokines of the different subclasses to displace 125I-IL-8 from GM 7373 cell surface was then explored. To this purpose, the C-X-C chemokines GROalpha /MGSA and neutrophil-activating peptide-2 and the C-C chemokines RANTES, MIP1alpha , and MIP1beta were utilized. As shown in Fig. 7B, 125I-IL-8 is similarly displaced by a 100-fold excess of unlabeled IL-8, GROalpha /MGSA, neutrophil-activating peptide-2, or RANTES, whereas MIP1alpha competes much less effectively and MIP1beta does not compete. Similar results were obtained when unlabeled chemokines were evaluated for their capacity to displace 125I-GROalpha /MGSA from GM 7373 cell surface (data not shown).


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Fig. 7.   Binding of 125I-IL-8 to GM 7373 cells. A, Scatchard plot analysis. GM 7373 cells (1 × 106 cells/ml) were incubated with 500 pM radiolabeled IL-8 in the presence of increasing concentrations of unlabeled IL-8 at 4 °C for 1 h. Binding was terminated by centrifugation through oil, and the cell pellets were counted. The Scatchard plot shown here is representative of three separate determinations. The binding shown represents specific binding. Nonspecific binding was around 5% of total 125I-IL-8 added and has been subtracted. B, competition binding studies. Cells were incubated for 1 h at 4 °C with 125I-IL-8 in the absence of cross-competing ligand (vehicle) or with 1 µM concentrations of unlabeled chemokines as shown. The binding reactions were stopped as described above. NAP, neutrophil-activating peptide-2.

The characteristics of the chemokine-binding sites (low affinity, high capacity, and promiscuity) suggest that cell surface HSPGs are likely involved in the interaction of IL-8 with GM 7373 cells. To assess this hypothesis, the effect of different free GAGs on IL-8 activity was investigated. To this purpose, GM 7373 cells were preincubated for 3 h at 37 °C with IL-8 in the presence of 100 ng/ml GAG under test. After extensive washing of the cell monolayers, cells were incubated for a further 24 h with bFGF and counted. As shown in Fig. 8A, heparin and to a lesser extent HS were able to abrogate the inhibitory activity of IL-8, whereas chondroitin-4-sulfate, hyaluronic acid, and K5 polysaccharide were ineffective. Also, total O-desulfation and selective 2-O-desulfation abolished the capacity of heparin to antagonize IL-8 activity, whereas 6-O- or N-desulfated heparins were only partially effective (Fig. 8B), suggesting that 2-O-sulfate groups are those mainly involved in IL-8-heparin interaction.


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Fig. 8.   Effect of different GAGs and selectively desulfated heparins on the inhibitory activity of IL-8. GM 7373 cells were incubated for 3 h at 37 °C with no GAG or with 100 ng/ml heparin (UFH), HS, chondroitin-4-sulfate (Ch4S), hyaluronic acid (HA), K5-polysaccharide (K5-PS) (in A) or totally O-desulfated (Tot-O-DS), 2-O-desulfated (2-O-DS), 6-O-desulfated (6-O-DS), or N-desulfated (N-DS) heparins (in B) in the absence (hatched bar) or in the presence (black bar) of 100 ng/ml IL-8. Cells were then washed extensively and incubated for a further 24 h with 10 ng/ml bFGF. At the end of the incubation, cells were trypsinized and counted. Data are expressed as percentages of the stimulation of cell proliferation exerted by bFGF alone when compared with untreated cultures. The results are the averages of triplicate wells, and similar results were obtained in two independent experiments.

IL-8 Inhibits bFGF Angiogenic Activity in Vivo-- The capacity of IL-8 to inhibit the mitogenic activity of bFGF in mouse SIECs isolated from neovascularized subcutaneous sponges (see Fig. 1) prompted us to assess whether the chemokine was also able to affect the angiogenic activity exerted in vivo by the growth factor in this experimental model. To this purpose, sponges were adsorbed with bFGF alone (100 ng/sponge) or with bFGF added with a molar excess of IL-8 (1 µg/sponge) and were implanted into the flank of anesthetized mice. After 7 days sponges were removed and neovascularization was compared with that measured in implants adsorbed with saline or with IL-8 alone. Despite a certain variability within experimental groups, the results indicate that IL-8 causes a significant decrease of the angiogenic activity of bFGF (Table II). Also, IL-8 alone does not exert a significant angiogenic response in this experimental model at the dose tested.

                              
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Table II
Effect of IL-8 on the angiogenic activity of bFGF
Sponges (approximately 1 cm3) containing saline, 100 ng of bFGF, 1 µg of IL-8, or bFGF plus IL-8 were inserted subcutaneously in the flank of anesthetized mice. After 7 days sponges were removed, and neovascularization was quantified by measuring their hemoglobin content.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This work demonstrates that IL-8 inhibits the mitogenic response elicited by bFGF on certain endothelial cell types and significantly affects its angiogenic activity in vivo. At variance with previous results on the inhibitory activity exerted by PF-4 on bFGF (51), several lines of evidence indicate that the mechanism of action of IL-8 is not competitive: (i) the inhibitory activity of IL-8 on cultured endothelial GM 7373 cells is not overcome by increasing concentrations of the growth factor; (ii) IL-8 does not compete for the binding of 125I-bFGF to high affinity FGFRs and low affinity HSPGs; and (iii) a short interaction of IL-8 with GM 7373 cells, followed by the removal of the chemokine before the addition of bFGF, is sufficient to prevent the mitogenic response. The demonstration that the inhibitory activity of IL-8 is abolished by the PKC inhibitors H-7 and GF 109 and mimicked by the PKC activator TPA points instead to a role for PKC in mediating IL-8 action on GM 7373 cells. To our knowledge, this is the first evidence for the chemokine-dependent activation of a signal transduction pathway that modulates the response of endothelial cells to angiogenic growth factor(s).

PKC activation has been demonstrated to attenuate signaling from tyrosine kinase growth factor receptors (48). We have found that IL-8 does not affect the capacity of bFGF to activate ERK-2, an early downstream signal that follows receptor tyrosine phosphorylation (34), thus indicating that IL-8 does not affect the tyrosine kinase activity of FGFR. However, IL-8 causes a approx 60% down-regulation of FGFRs that may explain, at least in part, the inhibitory action of the chemokine. The observation that a partial down-regulation of FGFRs is sufficient to abolish the capacity of the target cell to respond to bFGF is not surprising and may reflect the existence of threshold effects in the long term FGFR signaling required to sustain bFGF-induced cell proliferation (30). Similar conclusions were drawn for TPA-treated HUVECs in which a partial decrease in FGFR binding capacity caused a complete suppression of the mitogenic activity of acidic FGF (49), a member of the FGF family that shares with bFGF similar receptor binding and biological activity (2).

Previous observations had shown that ELR+ and ELR- C-X-C chemokines exert opposite effects on angiogenesis in vitro and in vivo (17). However, contradictory results about the chemokine action on endothelial cells exist. For instance, the responsiveness of HUVECs to IL-8 has been subject to conflicting reports, perhaps reflecting differences in culture conditions (19). In addition, the anti-angiogenic chemokine IP-10 was reported in one study to inhibit bFGF-induced proliferation in HUVECs (23) but did not affect their proliferation in another (52). The ELR+ C-X-C chemokines GROalpha /MGSA and GRObeta , but not GROgamma , have been shown to inhibit the mitogenic activity of bFGF in bovine capillary endothelial cells (53). In the same study GRObeta reduced bFGF-stimulated mouse corneal neovascularization and tumor growth. In contrast, a second study (17) has shown that GROalpha /MGSA, GRObeta , and GROgamma can stimulate chemotaxis in bovine capillary endothelial cell and neovascularization of the rat cornea (limited to GROalpha /MGSA). Others have failed to observe any activity of GROalpha /MGSA on endothelial cells (14).

Here we report that various ELR+ and ELR- C-X-C chemokines, as well as C-C chemokines, share with IL-8 the capacity to inhibit bFGF activity in GM 7373 cells. A promiscuous, low affinity, high capacity chemokine-binding site was identified on these cells by binding assays with 125I-IL-8, 125I-GROalpha /MGSA, and 125I-RANTES. These data, together with the ability of soluble heparin and HS to overcome the inhibitory activity of IL-8, suggest that cell surface HSPGs play an important role in mediating chemokine activity in GM 7373 cells. Similar conclusions were drawn for some chemokines on different endothelial cell types. For instance, IP-10 and PF-4, which share a specific HSPG-binding site in SV-40-transformed murine endothelial cells, cause inhibition of bFGF-induced proliferation in HUVECs that is abrogated by soluble heparin (23). Similarly, inhibition of bFGF activity by PF-4 was overcome by exogenous heparin in 3T3 fibroblasts (51). However, this was not the case for the bFGF antagonist action of GROalpha /MGSA and GRObeta on bovine capillary endothelial cells, for which heparin is ineffective (53).

IL-8 exerts half-maximal inhibition of bFGF mitogenic activity at concentrations equal to 1-3 nM (see Fig. 1). At these doses, approximately 5,000 HSPG-like binding sites/cell are occupied by the chemokine on GM 7373 cells (KD equal to 132 nM). By assuming a tight coupling for these receptors, as observed for instance for IL-1 receptor (54), IL-8 receptor occupancy may be adequate for signaling to occur. Northern blot analysis has shown that endothelial GM 7373 cells express the transmembrane HSPG syndecan-1.3 Four tyrosine residues are highly conserved in the C-terminal of all the members of the syndecan family, and one of them fits a consensus sequence for tyrosine phosphorylation (55). Recently, tyrosine phosphorylation of the intracellular domain of syndecan-1 by cytoplasmic tyrosine kinases has been described in intact cells (56), thus supporting the hypothesis of HSPG involvement in signal transduction. Accordingly, endothelial HSPGs have been directly implicated in mediating the inhibitory activity of PF-4 and IP-10 (23). On the other hand, HSPGs have been demonstrated to mediate the binding of various heparin-binding growth factors to their high affinity receptors, including bFGF, vascular endothelial growth factor, and hepatocyte growth factor (5, 57, 58), and to facilitate receptor dimerization and signal transduction (7). Thus, even though we failed to detect the expression of the C-X-C receptors IL8-RA and IL-8RB mRNAs in GM 7373 cells,2 we cannot rule out the hypothesis that IL-8-HSPG interaction may present the chemokine to an as yet uncharacterized high affinity promiscuous chemokine receptor present at very low abundance on the cell surface. This receptor, undetected in the Scatchard plot analysis because of the high capacity of HSPGs, could be responsible for PKC activation, leading to down-regulation of FGFRs and eventually to the inhibition of bFGF activity. In keeping with this hypothesis is the capacity of HS to potentiate receptor-dependent neutrophil responses to IL-8, including [Ca2+]i increase and chemotaxis (38). In both cases, our data point to a role for endothelial cell HSPGs in mediating the antagonist activity of IL-8.

Endothelial cells are characterized by a high degree of heterogeneity, depending on tissue type, vessel caliber, and animal species from which they originated (37, 59). IL-8 exerts a bFGF antagonist action in two out of the six endothelial cell types here investigated, thus supporting previous observations on the different capacity of endothelial cells of different origin to respond to certain cytokines (discussed in Ref. 59). At present, it is not possible to anticipate how and whether a certain endothelial cell type will respond to IL-8. This may represent a serious limitation for studies aimed to elucidate the role of modulators in angiogenesis where in vivo data are frequently compared with in vitro observations obtained with large vessel endothelium originating from different animal species. A typical example is represented by the comparison of the results obtained in chick embryo chorioallantoic membrane, rabbit cornea, and/or rat cornea angiogenesis assays to observations on cultured endothelium isolated from bovine aorta and/or human umbilical vein. Among the endothelial cell types studied, we have investigated the effect exerted by IL-8 on murine endothelial cells (SIECs) isolated from the same anatomical district utilized for in vivo angiogenesis assays. We have observed that IL-8 inhibits the mitogenic activity of bFGF in SIECs isolated from neovascularized subcutaneous sponge implants and significantly reduces the in vivo angiogenic response in these bFGF-adsorbed implants. These data indicate that the bFGF-stimulated murine endothelium of this anatomical district is susceptible to the inhibitory activity of IL-8 both in vitro and in vivo. In apparent contrast with our observations, IL-8 has been demonstrated to promote new blood vessel formation in different experimental models, including rat corneal pocket (15), rabbit corneal pocket (16), rat mesenteric window (60), and rat sponge (61) angiogenesis assays, even though dose-response experiments have shown that angiogenesis occurs only over a narrow range of doses of the chemokine (15, 60). More data on the effect of IL-8 on endothelial cells isolated from these anatomical sites and animal species are required to clarify whether the observed IL-8-induced neovascularization depends on a direct action of the chemokine on endothelium or on secondary effects consequent to inflammatory cell recruitment.

Recent observations have shown that heparin-HS interaction of various angiogenic growth factors depends on specific structural characteristics of the polysaccharide chain (62). Also, heparin-HS subpopulations with chemokine binding selectivity exist (13). We have found that, at variance with heparin and HS, chondroitin-4-sulfate and nonsulfated hyaluronic acid and K5-polysaccharide were unable to overcome IL-8 activity, suggesting that the backbone structure, sulfation, and the arrangement of the charges are of importance in determining the capacity of GAGs to bind IL-8 and to modulate its activity on endothelial GM 7373 cells. The more significant effect of selective 2-O-desulfation in preventing the anti-bFGF activity of IL-8, when compared with 6-O- and N-desulfation, confirms this hypothesis. Thus, chemokines may bind heparin-HS in a distinct and possibly specific manner. Even though the specific chemokine-binding sequence(s) may be hidden in heparin because of its high degree of sulfation, the high heterogeneity in HS structure may allow a more refined tailoring of selective binding regions in different endothelial cell types or under different cell culture conditions that may influence the biological activity and bioavailability of HS-binding chemokines, thus explaining some of the contradictory results about chemokine action on endothelial cells.

In conclusion, a complex interplay occurs among chemokines, angiogenic growth factors, their cognate receptors, and cell surface HSPGs. Our data suggest that the different, possibly contrasting effects of various chemokines on endothelial cells of different origin may depend both on the type of chemokine-binding sites expressed and on the interplay between the activated chemokine- and growth factor-dependent signal transduction pathways. The stable endothelial GM 7373 cell line utilized in the present study may represent a useful tool to elucidate this interplay.

    ACKNOWLEDGEMENTS

We thank Dr. M. Rusnati for 125I-bFGF binding studies and helpful discussion and Dr. S. Ramponi for the in vivo angiogenesis assays.

    FOOTNOTES

* This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (Special Project on Angiogenesis), by the Ministero Superiore della Sanità (AIDS and Oncology Projects), by the Centro por lo Studio del Trattamento dello Scompenso Cardiaco, and by MURST (Project Inflammation, Biology, and Clinics; to M. P. and A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: General Pathology, School of Medicine, via Valsabbina 19, 25123 Brescia, Italy. Tel.: 39-30-3715315; Fax: 39-30-3701157; E-mail: presta{at}med.unibs.it.

1 The abbreviations used are: bFGF, basic fibroblast growth factor; FGFR, tyrosine kinase FGF receptor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; GAG, glycosaminoglycan; FCS, fetal calf serum; PBS, phosphate-buffered saline; BAEC, adult bovine aortic endothelial cell; MBEC, mouse brain microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; SIEC, sponge-induced mouse endothelial cell; DARC, Duffy antigen/erythrocyte chemokine receptor; ERK-2, extracellular signal-regulated kinase-2; PKC, protein kinase C; IL-8, interleukin-8; PF-4, platelet factor-4; GRO, growth-related oncogene; MGSA, melanoma growth stimulating activity; IP-10, interferon-gamma -inducible protein-10; MIP, macrophage inflammatory protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; H-7, 1-(5-isoquinolynsulfonyl)-2-methylpiperazine; H-8, N-[2-methylamino)ethyl]-5-isoquinoline-sulfonamide; GF 109, bisindolylmaleimide GF 109203X; RANTES, regulated on activation normal T cell expressed and secreted; MEM, Eagle's minimal essential medium.

2 R. Horuk, unpublished observations.

3 M. Presta and P. Dell'Era, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Klagsbrun, M., and D'Amore, P. A. (1991) Annu. Rev. Physiol. 53, 217-239[CrossRef][Medline] [Order article via Infotrieve]
  2. Basilico, C., and Moscatelli, D. (1992) Adv. Cancer Res. 59, 115-165[Medline] [Order article via Infotrieve]
  3. Gualandris, A., Urbinati, C., Rusnati, M., Ziche, M., and Presta, M. (1994) J. Cell. Physiol. 161, 149-159[Medline] [Order article via Infotrieve]
  4. Johnson, D. E., and Williams, L. T. (1993) Adv. Cancer Res. 60, 1-41[Medline] [Order article via Infotrieve]
  5. Rusnati, M., and Presta, M. (1996) Int. J. Clin. Lab. Res. 26, 15-23[Medline] [Order article via Infotrieve]
  6. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991) Science 252, 1705-1708[Medline] [Order article via Infotrieve]
  7. Spivak-Krolzman, T., Lemmon, M. A., Dikic, I., Ladbury, J. E., Pinchasi, D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J., and Lax, I. (1994) Cell 79, 1015-1024[Medline] [Order article via Infotrieve]
  8. Baggiolini, M., Dewald, B., and Moser, B. (1994) Adv. Immunol. 55, 97-179[Medline] [Order article via Infotrieve]
  9. Mantovani, A., Bussolino, F., and Introna, M. (1997) Immunol. Today 18, 231-239[CrossRef][Medline] [Order article via Infotrieve]
  10. Kelner, G. S., Kennedy, J., Bacon, K. B., Kleyensteuber, S., Largaespada, D. A., Jenkins, N. A., Copeland, N. G., Bazan, J. F., Moore, K. W., Schall, T. J., and Zlotnik, A. (1994) Science 266, 1395-1399[Medline] [Order article via Infotrieve]
  11. Murphy, P. M. (1996) Cytokine Growth Factor Rev. 1, 47-64
  12. Horuk, R. (1994) Immunol. Today 15, 169-174[CrossRef][Medline] [Order article via Infotrieve]
  13. Witt, D. P., and Lander, A. D. (1994) Curr. Biol. 4, 394-400[Medline] [Order article via Infotrieve]
  14. Rot, A. (1992) Immunol. Today 13, 291-294[CrossRef][Medline] [Order article via Infotrieve]
  15. Koch, A. E., Polverini, P. J., Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner, V. M., Elner, S. G., and Strieter, R. M. (1992) Science 258, 1798-1801[Medline] [Order article via Infotrieve]
  16. Strieter, R. M., Kunkel, S. L., Elner, V. M., Martonyi, C. L., Koch, A. E., Polverini, P. J., and Elner, S. G. (1992) Am. J. Pathol. 141, 1279-1284[Abstract]
  17. Strieter, R. M., Polverini, P. J., Kunkel, S. L., Arenberg, D. A., Burdick, M. D., Kasper, J., Dzuiba, J., Van Damme, J., Walz, A., Marriot, D., Chan, S.-Y., Roczniak, S., and Shanafelt, A. B. (1995) J. Biol. Chem. 270, 27348-27357[Abstract/Free Full Text]
  18. Maione, T. E., Gray, G. S., Petro, J., Hunt, A. J., Donner, A. L., Bauer, S. I., Carson, H. F., and Sharpe, R. J. (1990) Science 247, 77-79[Medline] [Order article via Infotrieve]
  19. Koch, A. E., Polverini, P. J., Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner, V. M., Elner, S. G., and Strieter, R. M. (1995) Science 21, 447-448
  20. Peiper, S. C., Wang, Z., Neote, K., Martin, A. W., Showell, H. J., Conklyn, M. J., Ogborne, K., Hadley, T. J., Lu, Z., Hesselgesser, J., and Horuk, R. (1995) J. Exp. Med. 181, 1311-1317[Abstract]
  21. Schonbeck, U., Brandt, E., Petersen, F., Flad, H.-D., and Loppnow, H. L. (1995) J. Immunol. 154, 2375-2383[Abstract/Free Full Text]
  22. Petzelbauer, P., Watson, C. A., Pfau, S. E., and Pober, J. S. (1995) Cytokine 7, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  23. Luster, A. D., Greenberg, S. M., and Leder, P. (1995) J. Exp. Med. 182, 219-231[Abstract]
  24. Isacchi, A., Statuto, M., Chiesa, R., Bergonzoni, L., Rusnati, M., Sarmientos, P., Ragnotti, G., and Presta, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2628-2632[Abstract]
  25. Coltrini, D., Rusnati, M., Zoppetti, G., Oreste, P., Grazioli, G., Naggi, A., and Presta, M. (1994) Biochem. J. 303, 583-590[Medline] [Order article via Infotrieve]
  26. Grispan, J. B., Stephen, N. M., and Levine, E. M. (1983) J. Cell. Physiol. 114, 328-338[Medline] [Order article via Infotrieve]
  27. Bastaki, M., Nelli, E. E., Dell'Era, P., Rusnati, M., Molinari-Tosatti, M. P., Parolini, S., Auerbach, R., Ruco, L. P., Possati, L. P., and Presta, M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 454-464[Abstract/Free Full Text]
  28. Dong, Q. G., Bernasconi, S., Lostaglio, S., Wainstock De Calmanovici, R., Martin-Padura, I., Breviario, F., Garlanda, C., Ramponi, S., Mantovani, A., and Vecchi, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 1599-1604[Abstract/Free Full Text]
  29. Presta, M., Maier, J. A. M., and Ragnotti, G. (1989) J. Cell Biol. 109, 1877-1884[Abstract]
  30. Presta, M., Tiberio, L., Rusnati, M., Dell'Era, P., and Ragnotti, G. (1991) Mol. Biol. Cell. 2, 719-726
  31. Rusnati, M., Urbinati, C., and Presta, M. (1993) J. Cell. Physiol. 154, 152-161[Medline] [Order article via Infotrieve]
  32. Moscatelli, D. (1987) J. Cell. Physiol. 131, 123-130[Medline] [Order article via Infotrieve]
  33. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672
  34. Besser, D., Presta, M., and Nagamine, Y. (1995) Cell Growth Differ. 6, 1009-1017[Abstract]
  35. Neote, K., Darbonne, W., Ogez, J., Horuk, R., and Schall, T. J. (1993) J. Biol. Chem. 268, 12247-12249[Abstract/Free Full Text]
  36. Passaniti, A., Taylor, R. M., Pili, R., Guo, Y., Long, P. V., Haney, J. A., Pauly, R. R., Grant, D. S., and Martin, G. R. (1992) Lab. Invest. 67, 519-528[Medline] [Order article via Infotrieve]
  37. McCarthy, S. A., Kuzu, I., Gatter, K. C., and Bicknell, R. (1991) Trends Pharmacol. Sci. 12, 462-467[CrossRef][Medline] [Order article via Infotrieve]
  38. Webb, L. M. C., Ehrengruber, M. U., Clark-Lewis, I., Baggiolini, M., and Rot, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7158-7162[Abstract]
  39. Presta, M., Moscatelli, D., Joseph-Silverstein, J., and Rifkin, D. B. (1986) Mol. Cell. Biol. 6, 4060-4066[Medline] [Order article via Infotrieve]
  40. Coffey, R. J., Leof, E. B., Shipley, G. D., and Moses, H. L. (1987) J. Cell. Physiol. 132, 143-148[Medline] [Order article via Infotrieve]
  41. Moscatelli, D., and Quarto, N. (1989) J. Cell Biol. 109, 2519-2527[Abstract]
  42. Kimata, H., Fujimoto, M., Lindley, I., and Furusho, K. (1995) Biochem. Biophys. Res. Commun. 207, 1044-1050[CrossRef][Medline] [Order article via Infotrieve]
  43. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041[Medline] [Order article via Infotrieve]
  44. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781[Abstract/Free Full Text]
  45. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 5592-5595[Abstract/Free Full Text]
  46. Hill, T. D., Dean, N. M., Mordan, L. J., Lau, A. F., Kanemitsu, M. Y., and Boynton, A. L. (1990) Science 248, 1660-1663[Medline] [Order article via Infotrieve]
  47. Maher, P. A. (1993) J. Biol. Chem. 268, 4244-4249[Abstract/Free Full Text]
  48. Carpenter, G. (1987) Ann. Rev. Biochem. 56, 881-914[CrossRef][Medline] [Order article via Infotrieve]
  49. Hoshi, H., Kan, M., Mioh, H., Chen, J.-K., and McKeehan, W. L. (1988) FASEB J. 2, 2797-2800[Abstract/Free Full Text]
  50. Hesselgesser, J., Chitnis, C. E., Miller, L. H., Yansura, D. G., Simmons, L. C., Fairbrother, W. J., Kotts, C., Wirth, C., Gillece-Castro, B. L., and Horuk, R. (1995) J. Biol. Chem. 270, 11472-11476[Abstract/Free Full Text]
  51. Watson, J. B., Getzler, S. B., and Mosher, D. F. (1994) J. Clin. Invest. 94, 261-268[Medline] [Order article via Infotrieve]
  52. Angiolillo, A. L., Sgadari, C., Taub, D. D., Liao, F., Farber, J. M, Maheshwari, S., Kleinman, H. K., Reaman, G. H., and Tosato, G. (1995) J. Exp. Med. 182, 155-162[Abstract]
  53. Cao, Y., Chen, C., Weatherbee, J. A., Tsang, M., and Folkman, J. (1995) J. Exp. Med. 182, 2069-2072[Abstract]
  54. Rosoff, P. M., Savage, N., and Dinarello, C. A. (1988) Cell 54, 73-81[Medline] [Order article via Infotrieve]
  55. Bernfiled, M., Kokenyesi, R., Kato, M., Hinkens, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef]
  56. Reiland, J., Ott, V. L., Lebakken, C. S., Yeaman, C., McCarthy, J., and Rapraeger, A. C. (1996) Biochem. J. 319, 39-47[Medline] [Order article via Infotrieve]
  57. Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld, G. (1992) J. Biol. Chem. 267, 6093-6098[Abstract/Free Full Text]
  58. Lyon, M., and Gallagher, J. T. (1994) Biochem. Soc. Trans. 22, 365-370[Medline] [Order article via Infotrieve]
  59. Zetter, B. R. (1988) in Endothelial Cells (Ryan, U., ed), Vol. 2, pp. 63-80, CRC Press, Inc., Boca Raton, FL
  60. Norrby, K. (1996) Cell Prolif. 29, 315-323[Medline] [Order article via Infotrieve]
  61. Hu, D. E., Hori, Y., Presta, M., Gresham, G. A., and Fan, T. P. (1994) Inflammation 18, 45-58[Medline] [Order article via Infotrieve]
  62. Lindahl, U., and Kjellen, L. (1991) Thromb. Haemostasis 66, 44-48[Medline] [Order article via Infotrieve]


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