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J Biol Chem, Vol. 274, Issue 39, 27617-27622, September 24, 1999


Vascular Endothelial Growth Factor-C Stimulates the Migration and Proliferation of Kaposi's Sarcoma Cells*

Serena MarchiòDagger , Luca Primo, Marco Pagano§, Giorgio Palestro§, Adriana Albini, Tanja Veikkolaparallel , Ilaria Cascone**, Kari Alitaloparallel , and Federico BussolinoDagger Dagger

From the Institute for Cancer Research and Treatment (I.R.C.C.) and Department of Genetics, Biology, and Biochemistry, University of Torino Medical School, 10060 Candiolo, Italy, the § Department of Biomedical Sciences and Oncology, University of Torino Medical School, 10100 Torino, Italy, the  National Institute for Cancer Research and Center of Advanced Biotechnology, 16100 Genova, Italy, and the parallel  Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, SF-00014 Helsinki, Finland

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype.

In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration.

Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

KS1 is an intensely angiogenic, multifocal proliferative disease of possible vascular origin (1). It is particularly frequent and aggressive when associated with infection by human immunodeficiency virus-1 (2), in contrast to classical KS, which is rare and indolent (2-5). To date, the pathogenesis of KS is not completely understood, but in vivo and in vitro evidence indicates that this sarcoma probably develops from reactive, non-tumoral cells (6, 7) that become characteristically "spindle"-shaped and induce angiogenesis when stimulated by a variety of cytokines and growth factors, including interleukin-1 and -6, interferon-gamma , granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha , fibroblast growth factors, platelet-derived growth factor, chemokines, VEGF-A, transforming growth factors (reviewed in Ref. 8). In later stages of development, a cell clone may assume neoplastic features, subsequent to genotypic alterations, causing KS hyperplastic lesions to transform into real sarcomas (9, 10). A putative candidate for this transformation is human herpesvirus-8 (11).

The nature of KS spindle cells has been debated for a long time. Recent studies indicate that these cells are a heterogeneous population with three distinct phenotypes, one reminiscent of activated vascular and lymphatic endothelial cells, the other one of macrophagic and dendritic cells, the last characterized by the presence of mixed markers of macrophage and endothelial cells (12-17). Cytokines exert a major role in KS development, at least in the beginning of the disease. Early lesions are infiltrated by inflammatory cells, mostly CD8+ T cells and macrophages, producing a variety of inflammatory cytokines and chemokines, such as interferon-gamma , tumor necrosis factor-alpha , interleukin-2 (reviewed in Ref. 18), that induce normal cells to acquire the KS phenotype (19). Cytokines produced by inflammatory cells present in the lesions can also stimulate KS cells themselves to produce other soluble mediators, including angiogenic factors. These molecules dictate the progression of the lesion by autocrine and paracrine mechanisms, regulating cell recruitment and growth, with consequent angiogenesis and lesion formation (18). Among these molecules, VEGF-A has been recently demonstrated to be involved in angiogenesis associated with KS by acting through an autocrine pathway. VEGF-A is produced by KS cells and promotes their growth both in vivo and in vitro in nude mice (20, 21). VEGF-A is present in KS lesions with basic fibroblast growth factor, which synergize to promote vascular permeability angiogenesis and further KS lesion formation (22). VEGF-A is known as one of the first regulators of angiogenesis (reviewed in Refs. 23 and 24). The recent identification of proteins with high similarity to VEGF-A led to the designation of a vascular endothelial family of growth factors, consisting of five members to date. In addition to VEGF-A, this family includes placental growth factor (25), VEGF-B (26), VEGF-C, VEGF-D (27-29), and VEGF-E (30).

VEGF-C was originally cloned as the specific ligand for VEGFR-3/Flt-4, a tyrosine-kinase receptor structurally related to VEGFR-1/Flt-1 and VEGFR-2/KDR, though unable to bind VEGF-A (29). VEGF-C is produced as a 61-kDa precursor that undergoes progressive proteolytic maturation, increasing its affinity and activating properties toward VEGFR-3 (31). VEGF-C has been identified as a specific regulator of lymphatic endothelia. In situ hybridization experiments in the mouse embryo show that both VEGF-C and VEGFR-3 are expressed at sites of lymphatic vessel formation (32). In the skin of transgenic mice, the overexpression of VEGF-C has been shown to induce lymphatic endothelial cell proliferation and hyperplasia of the lymphatic vasculature (33). Furthermore, in chicken mature chorioallantoic membrane assay, VEGF-C selectively induces growth of lymphatic vessels (34). Targeted inactivation of the gene encoding for VEGFR-3 resulted in defective remodeling and maturation of primitive vascular structures and in cardiovascular failure, suggesting a role for the VEGF-C/VEGFR-3 pathway in the development of vascular system before the differentiation between blood and lymph vascular networks (35). Recent evidence suggests a role for VEGF-C also in blood vascular system. In vitro, VEGF-C exhibits a mitogenic and chemotactic effect on vascular endothelial cells, and induction of angiogenesis in vitro by VEGF-C has been reported recently (29, 36-38). A possible in vivo action of this factor in angiogenesis is now under question, as the expression of its specific receptor VEGFR-3 is barely detectable in mature blood vessels (37). However, the 21-kDa completely processed VEGF-C glycoprotein can also bind and activate VEGFR-2 (29), so that the biological activity of VEGF-C in endothelium could be mediated by this receptor. Recent studies in vivo showed that VEGF-C can promote angiogenesis in rabbit ischemic limbs (36) and induce mouse cornea neovascularization and blood vessel development in chicken embryo chorioallantoic membrane assay (37).

Because of the presence of VEGFR-3 in KS lesions in vivo (39), in this study we analyzed the biological effect of VEGF-C on KS IMM, a KS-derived cell line that is very similar to typical spindle cell cultures established from KS lesions (9). VEGF-C promotes KS IMM cells migration and proliferation by activating the tyrosine phosphorylation of VEGFR-2 and VEGFR-3. These effects are evident also on human endothelial cells, indicating that VEGF-C could promote KS lesions by regulating functions of both KS spindle cells and associated endothelial cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cultures-- HUVEC were isolated from umbilical cord vein by collagenase treatment as described previously (40) and used at passages 1-4. KS IMM cells were derived from a non-AIDS patient (9) and are immortalized without signs of senescence after more than 120 in vitro passages. This cell line shares common markers and similar biological behavior with typical KS spindle cells (9). Cells were grown on gelatin-coated plastic, in medium 199 supplemented with 20% heat-inactivated FCS, penicillin (100 units/ml), streptomycin (50 µg/ml), heparin (50 µg/ml), and bovine brain extract (100 µg/ml) (Life Technologies, Inc., Milano, Italy).

Reagents-- Delta NDelta C 156S is a mutated form of VEGF-C in which Cys-156 has been replaced with a Ser (31, 41). Recombinant mature form of human wild type (Delta NDelta C) and mutant (Delta NDelta C 156S) VEGF-C were expressed in Pichia pastoris yeast cells and purified as described previously (31, 41).

KS-like Lesion Formation by KS IMM Cells in Nude Mice-- Healthy 10-week-old athymic nu/nu male mice were obtained from Charles River Laboratories (Colnago, Italy). KS IMM cells (3 × 106) were inoculated subcutaneously into the lower back of mice in the presence of matrigel (200 µl) (Collaborative Research, Bedford, MA). After 4-7 days from the injection, specimens were taken from the lesional sites, paraffin-embedded, and VEGFR-3 expression was analyzed immunohistochemically.

Evaluation of VEGFR-3 Expression in KS and in Endothelium-- Paraffin-embedded sections of nude mice lesions were processed through a series of decreasing ethanol concentrations and heated in a microwave oven in 10 mM sodium citrate (pH 6.0) at 750 watts for 10 min. Adjacent 5 µm sections were incubated with blocking serum (bovine serum albumin 100 mg/ml in PBS) and then with anti-VEGFR-3 mAb (1.1 µg/ml) (39) for 30 min at 37 °C. Slides were stained by using the streptavidin/biotin system (Dako Immunoglobulins, Glostrup, Denmark).

To detect VEGFR-3 in culture of KS IMM cells and HUVEC, cells were grown to confluence on gelatin-coated coverslips and fixed for 10 min in 3% paraformaldehyde, 2% saccharose in PBS (pH 7.6). Fixed cells were incubated with blocking reagent (10% v/v FCS in PBS) and then with anti-VEGFR-3 mAb (1.1 µg/ml) (39) in a humid chamber for 30 min at 37 °C. Fluorescent staining was visualized with a fluorescein isothiocyanate-conjugated secondary rabbit anti-mouse Ab (Sigma) and fluorescence microscopy.

Cell Growth Assays-- To assay mitogenic activity, KS IMM cells and HUVEC were seeded in 48-well plates (104 cells/well) and allowed to attach for 24 h. The cells were then starved in M199 containing 1% FCS for 24 h (HUVEC) or 96 h (KS IMM). Delta NDelta C or Delta NDelta C 156S was added to the wells in medium containing 2.5% FCS at the indicated concentrations. All treatments were made in triplicate. Cells were fixed in 2.5% glutaraldehyde, stained with 0.1% crystal violet in 20% methanol, and solubilized in 10% acetic acid. Cell growth was evaluated by measuring absorbance at 590 nm in a microplate reader (Bio-Rad, model 3530). A calibration curve was set up with known numbers of cells and a linear correlation between absorbance and cell counts was established up to 1 × 105 cells.

Chemotaxis Assays-- The cell migration assay was performed using a 48-well microchemotaxis chamber (Neuroprobe, Plaesanton, CA). Polyvinylpyrrolidone-free polycarbonate filters (Nucleopore, Corning Costar Corp., Cambridge, MA) with a pore size of 5 µm were coated with 1% gelatin for 10 min at room temperature and equilibrated in M199 supplemented with 1% FCS (40). Indicated concentrations of Delta NDelta C or Delta NDelta C 156S were placed in the lower compartment of a Boyden chamber. Subconfluent cultures, which had been starved as above, were harvested in PBS (pH 7.4) with 10 mM EDTA, washed once in PBS, and resuspended in M199 containing 1% FCS at a final concentration of 2.5×106 cells/ml. After placing the filter between lower and upper chambers, 50 µl of the cell suspension were seeded in the upper compartment. Cells were allowed to migrate for 7 h at 37 °C in a humidified atmosphere with 5% CO2. The filter was then removed, and cells on the upper side were scraped off with a rubber policeman. Migrated cells were fixed in methanol, stained with Giemsa solution (Diff-Quick, Baxter Diagnostics, Rome, Italy) and counted from five random high power fields (magnitude × 100) in each well. Each experimental point was studied in triplicate.

The direction of VEGF-C-stimulated migration was evaluated in KS IMM cells using a checkerboard analysis (42). Increasing concentrations of Delta NDelta C were placed in both top and bottom wells of the Boyden chamber in order to establish positive, absent, and negative concentration gradients across the filter barrier. Directed locomotion, chemotaxis, is a response to a gradient of attractant; random stimulated migration, chemokinesis, is the response to attractant when no concentration gradient is present.

Immunoprecipitation and Western Blotting-- Subconfluent cultures were starved as above and then cells were stimulated with the indicated concentrations of Delta NDelta C or Delta NDelta C 156S for 15 min at room temperature. After three washes with cold PBS containing 1 mM sodium orthovanadate, cells were lysed for 20 min on ice in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM ZnCl2, and 1% Triton X-100 (Sigma) (w/v). Lysates (1 mg of total proteins) were incubated at 4 °C for 2 h with 100 µl of a 50% solution of protein A-Sepharose (Amersham Pharmacia Biotech, Rainham, Essex, United Kingdom) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and anti-VEGFR-2 (C-1158, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-VEGFR-3 Ab (43). Immunoprecipitates were washed four times with lysis buffer and analyzed by 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto a nylon membrane (polyvinylidene difluoride, Millipore Corp., Bedford, MA) and analyzed by immunoblotting with anti-phosphotyrosine mAb (Upstate Biotechnology, Inc., Lake Placid, NY). Staining was performed by a chemiluminescence assay (ECL, Amersham Pharmacia Biotech).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

KS-derived Cells Induce KS-like Lesions in Nude Mice-- To determine whether KS IMM cells retain the ability to induce lesions resembling KS, nude mice were subcutaneously inoculated with 3 × 106 cells mixed with matrigel. In all mice, a lesion developed at the site of inoculation within 3-4 days, reaching maximal size by day 7, when the mice were sacrificed. Histologically, the macroscopic lesions induced by KS IMM cells were similar to those induced by other KS-derived cells (44). The neoplasia consisted of round and spindle cells, with vascular structures and capillaries and some infiltrated inflammatory cells (Fig. 1). The tumoral cells had an abundant basophylic cytoplasm and a large nucleus with one or more nucleoli and fine chromatin.


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  		Fig. 1.   Expression of VEGFR-3 in KS IMM, in HUVEC, and in nude mice lesions induced by KS IMM cells. A and B, cells were grown to confluence, fixed, and stained with anti-VEGFR-3 mAb, revealed with a fluorescein isothiocyanate-conjugated secondary rabbit anti-mouse Ab. C and D, in vivo lesions induced after 7 days by 3 × 106 KS IMM cells injected with 200 µl of Matrigel. The neoplasia consisted of round and spindle cells, with vascular structures and capillaries (arrows). Tissue specimens were fixed, paraffin-embedded, and stained by hematoxylin and eosin (C) or by anti-VEGFR-3 mAb (D). (Magnitude × 40.)

Expression of VEGFR-3 in KS Cells in Vitro and in Vivo and in Endothelial Cells in Vitro-- We analyzed the presence of VEGFR-3 in cultured KS IMM cells and HUVEC by immunofluorescence using an anti-VEGFR-3 mAb. The staining for VEGFR-3 was prominent (Fig. 1, A and B), and a similar pattern was observed in KS IMM-derived lesions in nude mice, evaluated by immunohistochemistry with the same anti-VEGFR-3 mAb (Fig. 1D). A strong cytoplasmatic staining particularly pronounced in the Golgi apparatus was observed, probably due to the presence of the p175 VEGFR-3 precursor (31). Incubation with secondary Ab alone did not give a detectable staining (not shown).

VEGF-C Is Mitogen for KS IMM and HUVEC-- We tested the ability of VEGF-C to induce proliferation of KS IMM cells, using HUVEC as a control, since previous studies have shown a VEGF-C-induced mitogenic effect on endothelial cells (36). On both KS IMM cells and HUVEC, Delta NDelta C stimulated a dose-response effect, reaching maximal induction at 100 ng/ml (Fig. 2). The increment in cell number was about 150% in both cell types.


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  		Fig. 2.   VEGF-C is mitogen for KS IMM and HUVEC. Cells were treated with Delta NDelta C, diluted in cell culture medium containing 2.5% FCS to give the concentration of growth factor indicated. Control, cells incubated in medium with 2.5% FCS only added. Cell number was evaluated 48 h after addition of growth factors as described under "Experimental Procedures." The results are expressed as the mean ± 1 S.D. of three independent experiments done in triplicate. F, evaluated by one-way analysis of variance, was 58.04 (A) or 22.04 (B). The Student-Newman-Keuls test gave p < 0.05 for the following comparison: A, untreated cells versus cells stimulated with 1, 10, 50, and 100 ng/ml Delta NDelta C; cells stimulated with 50 ng/ml versus cells stimulated with 1 and 100 ng/ml Delta NDelta C; or cells stimulated with 100 ng/ml versus cells stimulated with 1, 10, and 50 ng/ml Delta NDelta C; B, untreated cells versus cells stimulated with 100 ng/ml Delta NDelta C or cells stimulated with 100 ng/ml Delta NDelta C versus cells stimulated with 1, 10, and 50 ng/ml Delta NDelta C.

VEGF-C Induces Migration of KS IMM and HUVEC-- The effect of VEGF-C on KS IMM cell migration was analyzed in the Boyden chamber assay. Delta NDelta C exhibited a motogenic dose-response effect on KS IMM cells and on HUVEC, used as a positive control (31, 36). The KS IMM cell migration induced by Delta NDelta C was more than 6-fold stronger when compared with untreated cells, while HUVEC were less responsive (Fig. 3). The checkerboard analysis demonstrated that VEGF-C also has a chemokinetic component (increasing random migration) as well as a chemotactic component (Table I).


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  		Fig. 3.   VEGF-C induces migration of KS IMM and HUVEC. Cell migration was measured by Boyden chamber technique as detailed under "Experimental Procedures." The results are expressed as the mean ± 1 S.D. of three independent experiments done in triplicate. F, evaluated by one-way analysis of variance, was 26.77 (A) or 22.77 (B). The Student-Newman-Keuls test gave p < 0.05 for the following comparison: A, untreated cells versus cells stimulated with 10 and 100 ng/ml Delta NDelta C or cells stimulated with 1 ng/ml versus cells stimulated with 10 and 100 ng/ml Delta NDelta C; B, untreated cells versus cells stimulated with 10 and 100 ng/ml Delta NDelta C or cells stimulated with 1 ng/ml Delta NDelta C versus cells stimulated with 10 and 100 ng/ml Delta NDelta C.

                              
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Table I
VEGF-C exhibits a chemokinetic effect on KS IMM cells
The nature of motogenic effect on KS IMM cells was analyzed by a checkerboard assay. Showed concentrations of Delta NDelta C were placed in both top and bottom wells of the Boyden chamber in order to establish positive, absent, and negative concentration gradients across the filter barrier. The results are expressed as the mean ± 1 S.D. of three independent experiments made in triplicate.

VEGF-C Induces Tyrosine Phosphorylation of VEGFR-2 and VEGFR-3-- VEGF-C has been demonstrated to activate both VEGFR-2 and VEGFR-3 tyrosine phosphorylation in NIH-3T3 and in porcine aortic endothelial cells respectively transfected with KDR and flt-4 cDNAs, which overexpress these proteins (29). To verify whether the biological responses observed in KS IMM cells and HUVEC were due to the activation of these receptors, tyrosine phosphorylation of VEGFR-2 and VEGFR-3 was analyzed. In a time course experiment, KS IMM cells were stimulated with 100 ng/ml of Delta NDelta C, a concentration sufficient to induce the mitogenic and motogenic response. Cell lysates were immunoprecipitated using a polyclonal anti-VEGFR-3 mAb, and proteins were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted by an anti-phosphotyrosine mAb. Fig. 4 shows that tyrosine phosphorylation of the p125 proteolitically processed active form of VEGFR-3 increases after a 5-min stimulation, reaching a plateau after 15 min. Also the p195 unprocessed form of the receptor appears to be phosphorylated after 15 min, as observed previously in NIH-3T3 cells transfected with flt-4 cDNA (29). Delta NDelta C was able to activate the tyrosine phosphorylation of VEGFR-3 in HUVEC as well as in KS IMM cells (Fig. 5). Also VEGFR-2 was phosphorylated in response to Delta NDelta C stimulation in both cell types (Fig. 5).


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  		Fig. 4.   Time course stimulation of VEGFR-3 in KS IMM. Cells were stimulated with Delta NDelta C (100 ng/ml), and after lysis the receptor was immunoprecipitated using a polyclonal anti-VEGFR-3 Ab and analyzed by Western blotting with an anti-phosphotyrosine mAb. Arrows denote the position of the phosphorylated proteolytically processed 125-kDa form and of the unprocessed 195-kDa form of VEGFR-3. This experiment is representative of three performed with similar results.


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  		Fig. 5.   VEGF-C induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3. KS IMM cells and HUVEC were incubated with Delta NDelta C (100 ng/ml) for 15 min. Cells were lysed, and receptors were immunoprecipitated with the specific anti-receptor Ab and analyzed by Western blotting with an anti-phosphotyrosine mAb. Upper panels, activation of VEGFR-3 by VEGF-C in KS IMM and HUVEC. Arrows denote the position of the phosphorylated proteolytically processed 125-kDa form and the unprocessed 195-kDa form of VEGFR-3. Lower panels, activation of VEGFR-2. Arrows denote the position of the phosphorylated 210-kDa VEGFR-2 protein. The figure is representative of one typical experiment out of three performed.

A Mutant Form of VEGF-C Unable to Bind VEGFR-2 Has Altered Biological Activities on KS IMM Cells-- To discriminate the functional roles of VEGFR-2 and VEGFR-3 in the biological responses to VEGF-C, we tested the effect of the VEGF-C mutant Delta NDelta C 156S on KS IMM cells. Delta NDelta C 156S is a mature form of VEGF-C in which Cys-156 has been replaced with a Ser. The mutated form is a selective ligand and an activator of VEGFR-3, while devoid of any VEGFR-2 activation property (31, 41). We first analyzed the tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in starved KS IMM cells stimulated with Delta NDelta C 156S (100 ng/ml). Immunoprecipitation with anti-VEGFR-2 and anti-VEGFR-3 Ab, followed by Western blotting with anti-phosphotyrosine mAb, shows that Delta NDelta C 156S clearly stimulated tyrosine phosphorylation of VEGFR-3, but not of VEGFR-2 (Fig. 6). The motogenic and mitogenic activities of Delta NDelta C 156S on KS IMM cells are shown in Fig. 7. The mutant VEGF-C, used at the concentration able to induce tyrosine phosphorylation of VEGFR-3, activates the migration but not the proliferation of KS IMM cells.


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  		Fig. 6.   Delta NDelta C 156S is a mutated mature VEGF-C form that selectively activates VEGFR-3. After stimulation with Delta NDelta C 156S (100 ng/ml) and Delta NDelta C (100 ng/ml) for 15 min, cells were lysed, and receptors were immunoprecipitated with anti-receptor Ab and analyzed by Western blotting with an anti-phosphotyrosine mAb. Arrows denote the position of the phosphorylated proteolytically processed 125-kDa form and the unprocessed 195-kDa form of VEGFR-3 (upper panel) and of the 210-kDa VEGFR-2 protein (lower panel). The figure is representative of one typical experiment out of three performed.


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  		Fig. 7.   Delta NDelta C 156S activates the migration but not the proliferation of KS IMM cells. KS IMM cells were treated with Delta NDelta C and Delta NDelta C 156S (100 ng/ml). Proliferation (A) and migration (B) were evaluated as detailed in the legend to Figs. 2 and 3, respectively. The results are expressed as the mean ± 1 S.D. of three independent experiments done in triplicate. F, evaluated by one-way analysis of variance, was 37.52 (A) or 211.91 (B). The Student-Newman-Keuls test gave p < 0.05 for the following comparison: A, control versus cells stimulated with Delta NDelta C; cells stimulated with Delta NDelta C versus cells stimulated with Delta NDelta C 156S; B, control versus cells stimulated with Delta NDelta C and with Delta NDelta C 156S or cells stimulated with Delta NDelta C versus cells stimulated with Delta NDelta C 156S.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two structurally related tyrosine kinase receptors, VEGFR-2 and VEGFR-3, have been reported to bind and to be activated by VEGF-C. First evidence indicated that the VEGF-C/VEGFR-3 pathway could be a specific regulator of lymphoangiogenesis (32, 33, 39, 45), but a role for VEGF-C in blood vascular endothelium is now emerging, as recent studies demonstrated a VEGF-C-mediated angiogenic activity both in vitro and in vivo (36, 37).

Because of the putative lymphoendothelial origin of some KS cells (13), this tumor has been previously evaluated for the expression of VEGFR-3. In KS lesions in vivo, spindle cells were found to express VEGFR-3 protein (39). Vessels surrounding and invading the tumor, but not capillaries in the tumor itself, expressed the receptor, suggesting that VEGF-C could have a bifunctional role, both acting on KS spindle cells and favoring the vascularization of the lesions.

By immunochemical and immunohistochemical techniques, in this study we show that cultured KS IMM cells express VEGFR-3 protein (Fig. 1A). Furthermore, this receptor is also present in the in vivo lesions induced in nude mice by these cells (Fig. 1D). KS IMM cells also express VEGFR-2 protein, as demonstrated by immunoprecipitation experiments with a specific antibody (Fig. 5), in agreement with a previous reverse transcription-polymerase chain reaction analysis of different KS (21). In endothelial cells, the expression of VEGFR-3 has been detected in vitro only at the mRNA level, by both reverse transcription-polymerase chain reaction (36, 46) and Northern blot (45). We demonstrate by immunofluorescence experiments that HUVEC express the VEGFR-3 protein (Fig. 1B).

The presence of VEGFR-2 and -3 on KS IMM suggests that this cell line has a mixed macrophage-endothelial phenotype as already shown in vivo in KS lesions (17). Actually, KS IMM does not express CD-144, CD-31, factor VIII, and CD-62E, but does express vimentin (9) and low amounts of smooth muscle alpha -actin.2 CD54 is positive, while CD-106 is expressed at low levels, but inducible upon treatment with tumor necrosis factor-alpha and interferon-gamma . Inflammatory cytokines also induce high levels of long pentraxin (9), a marker of monocyte and endothelial cells (47). KS IMM stimulated by interferon-gamma express major histocompatibility class II antigens, constitutively expressed on macrophages and in interferon-gamma -stimulated endothelial cells (9). Finally KS IMM express also the chemokine receptor CCR52 typically found on macrophages (48).

VEGF-C can act on KS IMM cells and on HUVEC by activating both VEGFR-2 and VEGFR-3 tyrosine phosphorylation and stimulating a biological effect that involves a mitogenic and motogenic response. VEGF-C activates tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in both cell types (Fig. 5). However, KS IMM cells are more responsive than HUVEC to the motogenic activation by VEGF-C (Fig. 3). At the moment, this discrepancy cannot be easily explained. It could be due to (i) the presence of possible co-receptors that modify the receptor affinity (49, 50), (ii) the expression of specific integrins that modulate the activity of angiogenic inducers (23), or (iii) a difference in intracellular signaling molecules or the possibility of a difference in the receptor expression levels.

The activation of VEGFR-2 and VEGFR-3 by VEGF-C was previously seen only in cells transfected with receptor cDNAs, which overexpress these proteins (29). Using porcine aortic endothelial cells selectively expressing VEGFR-2 or VEGFR-3, Cao and co-wokers (37) showed that VEGF-C can promote migration and proliferation independently signaling through one receptor only.

The activation of both VEGFR-2 and VEGFR-3 in KS IMM cells could suggest a functional redundancy or alternatively indicate different activities triggered by these two receptors. To discriminate between these two hypotheses, we tested the effect of the VEGF-C mutant Delta NDelta C 156S on KS IMM cells. Delta NDelta C 156S, a mutated mature form of VEGF-C in which Cys-156 has been replaced with a Ser, is a specific VEGFR-3 agonist, as it does not bind nor activate VEGFR-2 (31, 41). In KS IMM cells, Delta NDelta C 156S activates the tyrosine phosphorylation of VEGFR-3 only (Fig. 6), elicits a weak motogenic effect, and is devoid of mitogenic activity (Fig. 7). Our experiments demonstrate that VEGFR-3 is involved in inducing cell migration, while VEGFR-2 is essential in transducing a proliferative signal. The disagreement between our present data and previous results showing that VEGFR-2 and VEGFR-3 trigger overlapped biological signals (37) could be due to the different technical approach used. Previous results have been obtained in transfected cells, whereas we studied cells that constitutively express VEGFR-2 and VEGFR-3. Moreover, it would be interesting to evaluate the presence of possible VEGFR-2/VEGFR-3 heterodimers, probably correlated with the receptor expression levels. This hypothesis will be further investigated.

Recent data demonstrate that proinflammatory cytokines, such as interleukin-1alpha , interleukin-1beta , and tumor necrosis factor-alpha , are responsible of VEGF-C mRNA transcriptional up-regulation and post-transcriptional stabilization in endothelial cells (51). It is intriguing to speculate that the high levels of inflammatory cytokines present in KS lesions (18) can induce an increased production of VEGF-C, which in turn can be instrumental in controlling KS cell growth and/or migration and invasion as well as in inducing associated angiogenesis.

    FOOTNOTES

* This work was supported by grants from European Community (Biomed-2 Project: BMHL-CT96-0669), Italian Association for Cancer Research (A.I.R.C.), Istituto Superiore di Sanità (II AIDS Project; Program on Tumor Therapy), Centro Nazionale delle Ricerche (C.N.R., Progetto Finalizzato Biotecnologie), and Ministero dell'Università e della Ricerca Scientifica e Tecnologica (M.U.R.S.T., 60% and Programmi di Rilevante Interesse Nazionale-1998).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.

Dagger Supported by a grant from C.N.R. (Progetto Biotecnologie).

** Supported by a grant from Fondazione piemontese "Gigi Ghirotti."

Dagger Dagger To whom correspondence should be addressed: Institute for Cancer Research and Treatment (I.R.C.C.), Dept. of Genetics, Biology, and Biochemistry, University of Torino Medical School, s.p. 142, 10060 Candiolo, Italy. Tel.: 39-11-9933347; Fax: 39-11-9933524; E-mail: fbussoli@mail.ircc.unito.it.

2 A. Albini, unpublished data.

    ABBREVIATIONS

The abbreviations used are: KS, Kaposi's sarcoma; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; HUVEC, human umbilical vein endothelial cells; FCS, fetal calf serum; PBS, phosphate-buffered saline; Ab, antibody; mAb, monoclonal Ab.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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