Vascular Endothelial Growth Factor-C Stimulates the Migration and Proliferation of Kaposi's Sarcoma Cells
1999; Elsevier BV; Volume: 274; Issue: 39 Linguagem: Inglês
10.1074/jbc.274.39.27617
ISSN1083-351X
AutoresSerena Marchiò, Luca Primo, Marco Pagano, Giorgio Palestro, Adriana Albini, Tanja Veikkola, Ilaria Cascone, Kari Alitalo, Federico Bussolino,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoRecent 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. 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. Kaposi's sarcoma vascular endothelial growth factor VEGF receptor human umbilical vein endothelial cells fetal calf serum phosphate-buffered saline antibody monoclonal Ab KS1 is an intensely angiogenic, multifocal proliferative disease of possible vascular origin (1Ruszczak Z. Mayer-Da Silva A. Orfanos C.E. Am. J. Dermopathol. 1987; 9: 388-392Crossref PubMed Scopus (52) Google Scholar). It is particularly frequent and aggressive when associated with infection by human immunodeficiency virus-1 (2Gottlieb G.J. Ackerman A.B. Human Pathol. 1981; 13: 882-892Crossref Scopus (145) Google Scholar), in contrast to classical KS, which is rare and indolent (2Gottlieb G.J. Ackerman A.B. Human Pathol. 1981; 13: 882-892Crossref Scopus (145) Google Scholar, 3Penn I. Transplantation. 1979; 27: 8-11Crossref PubMed Scopus (292) Google Scholar, 4Slavin G. Cameron H.M. Singh H. Br. J. Cancer. 1969; 23: 349-357Crossref PubMed Scopus (68) Google Scholar, 5Taylor J.F. Smith P.G. Bull D. Pike M.C. Br. J. Cancer. 1972; 26: 483-497Crossref PubMed Scopus (77) Google Scholar). To date, the pathogenesis of KS is not completely understood, but in vivoand in vitro evidence indicates that this sarcoma probably develops from reactive, non-tumoral cells (6Ensoli B. Salahuddin S.Z. Gallo R.C. Cancer Cells. 1989; 1: 93-96PubMed Google Scholar, 7Brooks J.J. Lancet. 1986; 2: 1309-1311Abstract PubMed Scopus (120) Google Scholar) that become characteristically "spindle"-shaped and induce angiogenesis when stimulated by a variety of cytokines and growth factors, including interleukin-1 and -6, interferon-γ, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, fibroblast growth factors, platelet-derived growth factor, chemokines, VEGF-A, transforming growth factors (reviewed in Ref. 8Ensoli B. Barillari G. Gallo R.C. Immunol. Rev. 1992; 127: 147-155Crossref PubMed Scopus (134) Google Scholar). 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 (9Albini A. Paglieri I. Orengo G. Carlone S. Aluigi M.G. DeMarchi R. Matteucci C. Mantovani A. Carozzi F. Donini S. Benelli R. AIDS. 1997; 11: 713-721Crossref PubMed Scopus (103) Google Scholar, 10Lunardi-Iskandar Y. Gill P. Lam V.H. Zeman R.A. Michaels F. Mann D.L. Reitz M.S. Kaplan M. Berneman Z.N. Carter D. J. Natl. Cancer Inst. 1995; 87: 974-981Crossref PubMed Scopus (92) Google Scholar). A putative candidate for this transformation is human herpesvirus-8 (11Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar). 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 (12Dictor V. Andersson C. Am. J. Pathol. 1988; 130: 411-417PubMed Google Scholar, 13Beckstead J.H. Wood G.S. Fletcher V. Am. J. Pathol. 1985; 119: 294-300PubMed Google Scholar, 14Kaaya E.E. Parravicini C. Ordonez C. Gendelman R. Berti E. Gallo R.C. Biberfeld P. J. AIDS Hum. Retroviruses. 1995; 10: 295-305Crossref PubMed Google Scholar, 15Regezi S.A. MacPhail L.A. Daniels T.E. DeSouza Y.G. Greenspan J.S. Greenspan D. Am. J. Pathol. 1993; 143: 240-245PubMed Google Scholar, 16Zhang Y.M. Bachmann S. Hammer C. Van Lunzen D. Van Stemm A. Kern P. Fietrich M. Ziegler R. Waldmerr R. Nawroth P.P. Am. J. Pathol. 1994; 144: 51-59PubMed Google Scholar, 17Uccini S. Ruco L. Monardo F. Stopacciaro A. Dejana E. Lesnoni I. Cerimele D. Baroni C. J. Pathol. 1994; 173: 23-31Crossref PubMed Scopus (75) Google Scholar). 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-γ, tumor necrosis factor-α, interleukin-2 (reviewed in Ref. 18Ensoli B. Sturzl M. Cytokine Growth Factor Rev. 1998; 9: 63-83Crossref PubMed Scopus (164) Google Scholar), that induce normal cells to acquire the KS phenotype (19Fiorelli V. Gendelman R. Samaniego F. Markham P.D. Ensoli B. J. Clin. Invest. 1995; 95: 1723-1734Crossref PubMed Scopus (151) Google Scholar). 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 (18Ensoli B. Sturzl M. Cytokine Growth Factor Rev. 1998; 9: 63-83Crossref PubMed Scopus (164) Google Scholar). 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 (20Cornali E. Zietz C. Benelli R. Weninger W. Masiello L. Breier G. Tschaler E. Albini A. Sturzl M. Am. J. Pathol. 1996; 149: 1851-1869PubMed Google Scholar, 21Masood R. Cai J. Zheng T. Smith D.L. Naidu Y. Gill P.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar). VEGF-A is present in KS lesions with basic fibroblast growth factor, which synergize to promote vascular permeability angiogenesis and further KS lesion formation (22Samaniego F. Markham P.D. Gendelman R. Watanabe Y. Kao V. Kowalski K. Sonnabend J.A. Pintus A. Gallo R.C. Ensoli B. Am. J. Pathol. 1998; 152: 1433-1443PubMed Google Scholar). VEGF-A is known as one of the first regulators of angiogenesis (reviewed in Refs. 23Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (419) Google Scholar and 24Ferrara N. Davis-Smyth T. Endocrinol. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). 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 (25Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Del Vecchio S. Lei K.J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar), VEGF-B (26Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), VEGF-C, VEGF-D (27Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (265) Google Scholar, 28Yamada Y. Nezu J. Shimane M. Hirata Y. Genomics. 1997; 42: 483-488Crossref PubMed Scopus (229) Google Scholar, 29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar), and VEGF-E (30Ogawa S. Oku A. Sawano A. Yamaguchi S. Yazaki Y. Shibuya M. J. Biol. Chem. 1998; 273: 31273-31282Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 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 (29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). VEGF-C is produced as a 61-kDa precursor that undergoes progressive proteolytic maturation, increasing its affinity and activating properties toward VEGFR-3 (31Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (648) Google Scholar). 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 (32Kukk E. Lymboussaki A. Taira S. Kaipainen A. Jeltsch M. Joukov V. Development ( Camb. ). 1996; 122: 3829-3837PubMed Google Scholar). 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 (33Jeltsch M. Kaipanen A. Joukov V. Meng X. Lasko M. Rauvala H. Swartz M. Fukumura D. Jain R.K. Alitalo K. Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1114) Google Scholar). Furthermore, in chicken mature chorioallantoic membrane assay, VEGF-C selectively induces growth of lymphatic vessels (34Oh S.J. Jeltsch M.M. Birkenhager R. McCarty J.E. Weich H.A. Christ B. Alitalo K. Wiltig J. Dev. Biol. 1997; 199: 96-109Crossref Scopus (433) Google Scholar). 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 (35Dumont D.J. Jussila L. Taipale J. Lymboussaki A. Mustonen T. Pajusola K. Breitman M. Alitalo K. Science. 1998; 282: 946-949Crossref PubMed Scopus (699) Google Scholar). 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 (29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar, 36Witzenbichler B. Asahara T. Murohara T. Silver M. Spyridopoulos I. Magner M. Principe N. Kearney M. Hu J.S. Isner J.M. Am. J. Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 37Cao Y. Linden P. Farnebo J. Cao R. Eriksno A. Kumar V. Qi J. Claesson-Welsh L. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14389-14394Crossref PubMed Scopus (496) Google Scholar, 38Pepper M.S. Mandriota S.J. Jeltsch M. Kumar V. Alitalo K. J. Cell. Physiol. 1998; 177: 439-452Crossref PubMed Scopus (154) Google Scholar). 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 (37Cao Y. Linden P. Farnebo J. Cao R. Eriksno A. Kumar V. Qi J. Claesson-Welsh L. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14389-14394Crossref PubMed Scopus (496) Google Scholar). However, the 21-kDa completely processed VEGF-C glycoprotein can also bind and activate VEGFR-2 (29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar), 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 (36Witzenbichler B. Asahara T. Murohara T. Silver M. Spyridopoulos I. Magner M. Principe N. Kearney M. Hu J.S. Isner J.M. Am. J. Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar) and induce mouse cornea neovascularization and blood vessel development in chicken embryo chorioallantoic membrane assay (37Cao Y. Linden P. Farnebo J. Cao R. Eriksno A. Kumar V. Qi J. Claesson-Welsh L. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14389-14394Crossref PubMed Scopus (496) Google Scholar). Because of the presence of VEGFR-3 in KS lesions in vivo(39Jussila L. Valtola R. Partanen T.A. Salven P. Heikkila P. Matikainen M.T. Renkonen R. Kaipanen A. Detmar M. Tschachler E. Alitalo R. Alitalo K. Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar), 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 (9Albini A. Paglieri I. Orengo G. Carlone S. Aluigi M.G. DeMarchi R. Matteucci C. Mantovani A. Carozzi F. Donini S. Benelli R. AIDS. 1997; 11: 713-721Crossref PubMed Scopus (103) Google Scholar). 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. HUVEC were isolated from umbilical cord vein by collagenase treatment as described previously (40Bussolino F. Arese M. Montrucchio G. Barra L. Primo L. Benelli R. Sanavio F. Aglietta M. Ghigo D. Rola-Pleszczynski M.R. Albini A. Camussi G. J. Clin. Invest. 1995; 96: 940-952Crossref PubMed Scopus (92) Google Scholar) and used at passages 1–4. KS IMM cells were derived from a non-AIDS patient (9Albini A. Paglieri I. Orengo G. Carlone S. Aluigi M.G. DeMarchi R. Matteucci C. Mantovani A. Carozzi F. Donini S. Benelli R. AIDS. 1997; 11: 713-721Crossref PubMed Scopus (103) Google Scholar) and are immortalized without signs of senescence after more than 120in vitro passages. This cell line shares common markers and similar biological behavior with typical KS spindle cells (9Albini A. Paglieri I. Orengo G. Carlone S. Aluigi M.G. DeMarchi R. Matteucci C. Mantovani A. Carozzi F. Donini S. Benelli R. AIDS. 1997; 11: 713-721Crossref PubMed Scopus (103) Google Scholar). 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). ΔNΔC 156S is a mutated form of VEGF-C in which Cys-156 has been replaced with a Ser (31Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (648) Google Scholar, 41Joukov V. Kumar V. Sorsa T. Arighi E. Weich H. Saksela O. Alitalo K. J. Biol. Chem. 1998; 273: 6599-6602Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Recombinant mature form of human wild type (ΔNΔC) and mutant (ΔNΔC 156S) VEGF-C were expressed in Pichia pastoris yeast cells and purified as described previously (31Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (648) Google Scholar, 41Joukov V. Kumar V. Sorsa T. Arighi E. Weich H. Saksela O. Alitalo K. J. Biol. Chem. 1998; 273: 6599-6602Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). 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. 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) (39Jussila L. Valtola R. Partanen T.A. Salven P. Heikkila P. Matikainen M.T. Renkonen R. Kaipanen A. Detmar M. Tschachler E. Alitalo R. Alitalo K. Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar) 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) (39Jussila L. Valtola R. Partanen T.A. Salven P. Heikkila P. Matikainen M.T. Renkonen R. Kaipanen A. Detmar M. Tschachler E. Alitalo R. Alitalo K. Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar) 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. To assay mitogenic activity, KS IMM cells and HUVEC were seeded in 48-well plates (104cells/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). ΔNΔC or ΔNΔC 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. 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 (40Bussolino F. Arese M. Montrucchio G. Barra L. Primo L. Benelli R. Sanavio F. Aglietta M. Ghigo D. Rola-Pleszczynski M.R. Albini A. Camussi G. J. Clin. Invest. 1995; 96: 940-952Crossref PubMed Scopus (92) Google Scholar). Indicated concentrations of ΔNΔC or ΔNΔC 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 (42Zigmond S.H. Hirsch J.G. J. Exp. Med. 1973; 137: 387-400Crossref PubMed Scopus (1280) Google Scholar). Increasing concentrations of ΔNΔC 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. Subconfluent cultures were starved as above and then cells were stimulated with the indicated concentrations of ΔNΔC or ΔNΔC 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 mmphenylmethylsulfonyl 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 (43Pajusola K. Aprelikova O. Pelicci G. Weich H. Claesson-Welsh L. Alitalo K. Oncogene. 1994; 9: 3545-3555PubMed Google Scholar). 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). 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 (44Ensoli B. Gendelman R. Markham P. Fiorelli V. Colombini S. Raffeld M. Cafaro A. Chang H.K. Brady J.N. Gallo R.C. Nature. 1994; 371: 674-680Crossref PubMed Scopus (542) Google Scholar). 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. 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. 1 D). A strong cytoplasmatic staining particularly pronounced in the Golgi apparatus was observed, probably due to the presence of the p175 VEGFR-3 precursor (31Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (648) Google Scholar). Incubation with secondary Ab alone did not give a detectable staining (not shown). 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 (36Witzenbichler B. Asahara T. Murohara T. Silver M. Spyridopoulos I. Magner M. Principe N. Kearney M. Hu J.S. Isner J.M. Am. J. Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). On both KS IMM cells and HUVEC, ΔNΔC 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. The effect of VEGF-C on KS IMM cell migration was analyzed in the Boyden chamber assay. ΔNΔC exhibited a motogenic dose-response effect on KS IMM cells and on HUVEC, used as a positive control (31Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (648) Google Scholar, 36Witzenbichler B. Asahara T. Murohara T. Silver M. Spyridopoulos I. Magner M. Principe N. Kearney M. Hu J.S. Isner J.M. Am. J. Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The KS IMM cell migration induced by ΔNΔC 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 (TableI).Table IVEGF-C exhibits a chemokinetic effect on KS IMM cellsLower chamber concentrationsUpper chamber concentrationsControl10 ng/ml30 ng/mlControl5.40 ± 0.6010.80 ± 0.4414.00 ± 0.7410 ng/ml11.40 ± 0.6012.90 ± 0.7815.40 ± 0.6030 ng/ml15.00 ± 1.0820.20 ± 0.8622.60 ± 0.94a Migrated cells per fields. Open table in a new tab a Migrated cells per fields. 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 andflt-4 cDNAs, which overexpress these proteins (29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). 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 ΔNΔC, 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 (29Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). ΔNΔC 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 ΔNΔC stimulation in both cell types (Fig. 5).Figure 5VEGF-C induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3. KS IMM cells and HUVEC were incubated with ΔNΔC (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-phospho
Referência(s)