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© The Rockefeller University Press,
0021-9525/1997//1375 $5.00
The Journal of Cell Biology, Volume 136, Number 6,
, 1997 1375-1384
Article |
Transferrin Promotes Endothelial Cell Migration and Invasion: Implication in Cartilage Neovascularization
Dipartimento di Oncologia Clinica e Sperimentale, Università di Genova, Genova, Italy; Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, Napoli, Italy; and || Istituto di Anatomia Umana Normale, Istologia ed Embriologia, Università di Bari, Bari, Italy
During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50–70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin.
The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium.
Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.
Abbreviations used in this paper: bFGF, basic FGF; CAM, choriallantoic membrane; CM, conditioned medium; KS, Kaposi sarcoma.
The regulation of angiogenesis is a key feature in many developmental and pathological processes (Folkman and Shing, 1992). Tumor angiogenesis, where resting endothelial cells are stimulated to proliferate, migrate, invade, and differentiate to form new vessels, is now considered a key target for controlling tumor growth and metastasis (Folkman, 1995). Growth plate cartilage is a very interesting tissue to investigate mechanisms controlling angiogenesis. Cartilage is normally an avascular tissue, but, after differentiation to hypertrophic cartilage in the growth plate, invasion by blood vessels is absolutely required for bone formation to proceed. Angiogenic inhibitors have been purified from both resting and proliferative cartilage (Moses et al., 1990, 1992; Pepper et al., 1991). In contrast, angiogenic activity has been described to be associated with mineralized hypertrophic chondrocytes in vitro (Brown and McFarland, 1992). We have recently shown (Descalzi Cancedda et al., 1995) that chick embryo chondrocytes undergoing in vitro differentiation from prechondrogenic to hypertrophic cells in the absence of ascorbic acid constitutively express antiangiogenic activity. However, when the hypertrophic chondrocyte culture is supplemented with ascorbic acid, there exists formation of a mineralized tissue in vitro similar to that found in vivo in calcified hypertrophic cartilage, and a switch to production of angiogenic activity. Therefore, the interaction of the cells with the surrounding organized extracellular matrix is an absolute requirement for the expression of the angiogenic activity.
We have also shown that in vitro differentiated hypertrophic chondrocytes cultured in the presence of ascorbic acid synthesize a large amount of a 69-kD protein identified as transferrin (Gentili et al., 1993). The presence of transferrin in vivo was demonstrated by immunohistochemistry and by in situ hybridization in the embryonic hypertrophic cartilage. The presence of the transferrin receptor on the surface of hypertrophic chondrocytes and, at particularly high levels, on the surface of adjacent undifferentiated cells in the diaphyseal collar was demonstrated. Based on these data, a possible autocrine and paracrine role of the transferrin in the developing bone was suggested (Gentili et al., 1994). Transferrins are a heterogeneous group of proteins caused by variations in the polypeptide chain, glycanation, and iron content. The major role of transferrins is iron transport; however, transferrin has other biological activities and has been found to be important for growth and differentiation of several cell types. These activities include a role as a mitogenic (Trowbridge and Omary, 1981), neurotrophic (Aizenman et al., 1986), and myotrophic (Li et al., 1982) factor as well as embryo morphogenesis (Ekblom et al., 1983; Partanen et al., 1984). In addition, a paracrine action of transferrin has also been shown in studies investigating the growth of metastatic tumors. The transferrin released by lung cells stimulates the growth of lung metastasizing tumor cells (Cavanaugh and Nicolson, 1991). The transferrin produced by brain cells plays a major role in supporting proliferation of melanoma cells in brain metastases (Menter et al., 1995).
Since the expression of transferrin in differentiating chondrocytes correlates with the switch to an angiogenicinducing phenotype, we have tested the hypothesis of a paracrine action of the transferrin in promoting neovascularization of growth plate cartilage.
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Alternatively, cells were cultured in a serum-free medium (Quarto et al., 1992). Dedifferentiated cells were suspended in Coon's modified F12 medium supplemented with T3 (10–10 M), insulin (60 ng/ml), and Dex (10–10 M). Fully differentiated hypertrophic chondrocytes were obtained after 1 wk in culture.
AIDS-KS cells (Albini et al., 1992) were cultured in RPMI 1640/DME (1:1) supplemented with 10% FCS (Gibco Ltd., Paisley, Scotland). EAhy 926 hybrid endothelial cells were the kind gift of Dr. C.J. Edgell (University of North Carolina, Chapel Hill) and were maintained in DME containing 10% FCS (Edgell et al., 1983).
Preparation of Conditioned Medium from Cultured Chondrocytes and Kaposi Sarcoma Cells
Hypertrophic chondrocytes in different culture conditions were rinsed twice with PBS and maintained in F12 medium for 24 h (5 ml for 100-mm dish) without any addition. This medium was collected and used for chemotaxis assay. Metabolic labeling of the cell culture after incubation with F12 alone confirmed that the protein synthesis remained unchanged in quality and quantity with respect to the previous culture conditions.
As positive controls for angiogenesis, conditioned media from Kaposi sarcoma (KS)1 cells were used (Albini et al., 1992, 1994; Salahuddin et al., 1988; Thompson et al., 1991). KS cells at 80% confluency were rinsed twice and incubated for 24 h with serum-free medium (8 ml per T75 flask) before conditioned media (CM) collection (KS-CM).
Conditioned media from both cultures were centrifuged for 10 min at 3,000 rpm to eliminate cell debris and stored at –20°C.
DNA Measurement
DNA content of cultured cells was determined to insure that corresponding concentrations of conditioned media were used. Cell layers were collected in 0.01% SDS in PBS and digested overnight at 50°C with proteinase K (Sigma Chemical Co., St. Louis, MO) at a concentration of 100 µg/ml in 10 mM Tris-HCl, pH 7.8, and 5 mM EDTA.
0.1 ml of each sample was added to 2 ml dye solution (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.1 M NaCl, and 0.1 mg/ml Hoechst 33258 [Sigma Chemical Co.], and DNA determination was performed in a DNA fluorimeter from Hoefer Scientific Instruments (San Francisco, CA).
Chemoinvasion Assay
The Boyden chamber chemoinvasion assay was carried out with slight modifications, as previously described (Albini et al., 1987). Matrigel, a mixture of basement membrane components obtained as an EnglebrethHolm-Swarm murine sarcoma extract (Kleinman et al., 1986), was layered evenly on polycarbonate filters (12-µm pore size, PVP-free; Nucleopore, Concorezzo, Italy) at a concentration of 20–25 µg per filter, air dried, and then reconstituted with medium into a solid gel.
Conditioned media from KS cells or chondrocytes were placed in the lower compartment of Boyden chamber and used as chemoattractants at a concentration proportional to the DNA content of cell layers.
In some experiments, the chemoattractant was transferrin from various sources: apotransferrin from human plasma (Sigma Chemical Co.), ovotransferrin (conalbumin) from chicken egg white (Sigma Chemical Co.), and ovotransferrin purified from chicken chondrocyte-conditioned medium, as previously described (Gentili et al., 1993). Conditioned media used in the different experiments presented in Results were derived from different primary cultures of hypertrophic chondrocytes.
We have previously demonstrated that EAhy 926 hybrid endothelial cells and human umbilical vein endothelial cells behaved similarly when the chemotactic activity of unfractionated medium conditioned by hypertrophic chondrocytes was tested (Descalzi Cancedda et al., 1995). Based on these results, EAhy 926 hybrid endothelial cells were used for the experiments reported in the present work. Endothelial cells (1.3 x 105 cells per chamber) were placed in the Boyden chamber's upper compartment after harvesting with trypsin and washing with serum-free DME.
Standard assay conditions were 6 h at 37°C in 5% CO2. After mechanical removal of the cells remaining on the upper surface of the filter, cells that migrated to the undersurface were stained (Toluidine blue; Sigma Chemical Co.) and quantitated microscopically. Five to ten random fields were counted on each filter. Assays were performed in triplicate and repeated at least twice. When antibodies were used, chemoattractants with and without antibodies and controls were previously incubated overnight at 4°C.
Chemotaxis Assay
Chemotaxis assays were performed in the same way as those of chemoinvasion, with the only difference being that the filters were not coated with Matrigel but with gelatin (type A; Sigma Chemical Co.) at a concentration of 5 µg/ml as an attachment substrate.
In some experiments, chemoattractants (conditioned media or transferrins) were mixed with the cells in the upper compartment (checkerboard analysis).
When antibodies were used, chemoattractants with and without antibodies and controls were previously incubated overnight at 4°C.
Cell Metabolic Labeling and SDS-PAGE Analysis of Proteins
Cells were labeled with [35S]methionine, as described by Descalzi Cancedda et al. (1992). Briefly, cultured cells were washed in PBS and incubated for 2 h at 37°C in methionine-free Coon's modified F12 medium with 100 µg/ml ascorbic acid. [35S]Methionine was then added and incubation continued for an additional 2 h. Aliquots of culture media were run for protein analysis on 15% SDS-PAGE in nonreducing conditions. Immunoprecipitation of specific proteins was performed as previously described (Descalzi Cancedda et al., 1992).
Western Blot Analysis
Cell layers were lysed with 0.1% SDS in PBS. Aliquots of samples were loaded on a 15% SDS polyacrylamide gel and electrophoresis was performed in reducing conditions. After electrophoresis, the gel was blotted to a BA85 nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany) according to the procedure described by Towbin et al. (1979).
The blot was saturated overnight with 5% skim milk powder (Merck Biochemica, Darmstadt, Germany) in TTBS buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween-20) and washed several times with TTBS; it was then incubated (for 3 h at room temperature) with the goat polyclonal antibody directed against the human placental transferrin receptor, made monospecific by adsorption on transferrin to avoid cross-reactivity (a gift from Dr. H.H. Sussman, Stanford University School of Medicine, Stanford, CA).
After further washes, detection was performed by a biotin-conjugated anti–goat IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and avidin-HRP (Jackson Immunoresearch Laboratories, Inc.), using 4-chloro-1-naphthol (Merck Biochemica) as substrate.
Chorioallantoic Membrane Assay
Fertilized White Leghorn chicken eggs (30 per group) were incubated under conditions of constant humidity at a temperature of 37°C. On the third day of incubation, a square window was opened in the egg shell after removal of 2–3 ml of albumen so as to detach the developing chorioallantoic membrane from the shell. The window was sealed with a glass of the same dimension, and the eggs were returned to the incubator.
Embryonic chorioallantoic membranes (CAMs) were treated at day 8 with (a) ovotransferrin purified from chicken hypertrophic chondrocyteconditioned medium (1 µg); (b) ovotransferrin from chicken egg white (Sigma Chemical Co.) (1 µg); (c) apotransferrin from human plasma (Sigma Chemical Co.) (1 µg); and (d) concentrated (3 µl) and not concentrated (3 µl) conditioned medium from cultured hypertrophic chondrocytes adsorbed on 1-mm3 sterilized gelatin sponge (Gelfoam; Upjohn Company, Kalamazoo, MI). Sponges were implanted on top of growing CAM under sterile conditions, within a laminar-flow hood. Sponges containing vehicle alone (PBS) and a stimulator of blood vessel formation, namely basic FGF (bFGF; 1 µg), were used as negative and positive controls, respectively.
CAMs were examined daily until day 12 and photographed in ovo under a stereomicroscope SR equipped with the Camera System MC 63 (Zeiss, Oberkochen, Germany). At day 12, CAMs were processed for light microscopy. Briefly, the embryos and their membranes were fixed in ovo in Bouin's fluid. The sponges and the underlying and immediately adjacent CAM portions were removed and processed for embedding in paraffin. 8-µm serial sections were cut according to a plane parallel to the surface of the CAM and stained with a 0.5% aqueous solution of Toluidine blue (Merck Biochemica) and observed under a Leitz-Dialux 20 light microscope (Leitz, Wetzlar, Germany).
At day 12, some CAMs were also injected intravascularly with India ink according to Olivo et al. (1992).
Quantitation of the Angiogenic Response
The angiogenic response was assessed by a planimetric method of point counting (Elias and Hyde, 1983; Ribatti et al., 1995). Briefly, every third section within 30 serial slides from an individual specimen was analyzed by a 144-point mesh inserted in the eyepiece of a Leitz-Dialux 20 photomicroscope. Six randomly chosen microscopic fields of each section were evaluated at x250 magnification for the total number of the intersection points that were occupied by vessels transversally cut (diameter ranging from 3–10 µm). Mean values ± SD for vessel counts were determined for each analysis.
The vascular density was indicated by the final mean number of the occupied intersection points. The statistical significance of differences between the mean values of the intersection points in the experimental CAMs and control ones was determined by the t test for unpaired data.
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In Vivo Assay (CAM) of the Angiogenic Activity of Transferrins and Hypertrophic Chondrocyte-conditioned Culture Medium
Angiogenic effects of transferrins of various origin and of hypertrophic chondrocyte-conditioned culture medium were investigated in vivo in the CAM assay. Embryonic chorioallantoic membranes at day 8 of incubation were implanted with sponges containing either ovotransferrin purified from chicken hypertrophic chondrocyte-conditioned medium, ovotransferrin from chicken egg white, apotransferrin from human plasma, or conditioned medium from cultured hypertrophic chondrocytes. Vehicle alone (PBS) and bFGF were used as negative and positive controls.
After 12 d of incubation, macroscopically, the majority of the sponges were surrounded by allantoic vessels (Table III); vessels were more numerous in the sponges treated with purified ovotransferrin and developed radially toward the implant in a "spoked wheel" pattern (Fig. 8, A and C). In the specimens treated with PBS, no vascular reaction was detectable around the sponges (Fig. 8, B and D).
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It is generally believed that cartilage chondrocytes produce migration and growth inhibitors for endothelial cells able to prevent blood vessel invasion even in the presence of angiogenic stimulatory factors. Angiogenic inhibitors have been purified from both resting and proliferative cartilage (Moses et al., 1990, 1992; Pepper et al., 1991). A suppressed synthesis of these antiangiogenic factors together with an activated synthesis of angiogenic factors in hypertrophic chondrocytes could explain the invasion of hypertrophic cartilage by blood vessels. An angiogenic activity has been described to be associated with mineralized hypertrophic chondrocytes in vitro (Brown and McFarland, 1992; Descalzi Cancedda et al., 1995).
We have demonstrated the high levels of ovotransferrin synthesis by hypertrophic chondrocytes both in vitro and in vivo (Gentili et al., 1993, 1994). Transferrins are monomeric, glycosylated, 80-kD proteins reversibly binding iron (de Jong et al., 1990). Hepatocytes are the main producer cells of this factor, but others such as Sertoli (Skinner et al., 1989), oviduct (McKnight et al., 1980), ependymal (Tsutsumi et al., 1989), and oligodendroglial (Bloch et al., 1985) cells synthesize significant amounts of transferrin.
In addition, transferrin differentially stimulates the growth of metastatic variant lines of murine melanoma (Nicolson et al., 1990) and human breast cancer cell lines (Inoue et al., 1993). Transferrin binding, but not growth response, correlates with invasive and metastatic properties of cell lines. This suggests that the transferrin/receptor system may be involved in more than just the support of cell proliferation. Endothelial cells have transferrin receptors (Jefferies et al., 1984) that are involved in transcytosis (Bickel et al., 1994); analogous to other cell types, these receptors could also be involved in endothelial cell proliferation and migration.
In the present study we have shown that transferrin is a chemotactic factor promoting migration and basement membrane invasion by endothelial cells. In the assay used to demonstrate this activity, endothelial cell migration was abolished by polyclonal antibodies directed against transferrin or by blocking antibodies directed against the transferrin receptor. We have previously shown a strong chemotactic and chemoinvasive activity, measured in Boyden chamber assays, in culture medium conditioned by hypertrophic chondrocytes (Descalzi Cancedda et al., 1995). In the presence of the antibodies against transferrin or the transferrin receptor, this activity decreased to 30–50% of the control. This finding indicates that transferrin is a major, but not the only, factor promoting cell migration in hypertrophic chondrocyte-conditioned culture medium and presumably in hypertrophic cartilage in vivo. Taking advantage of a serum-free culture system developed in our laboratory, we have shown that the medium conditioned by hypertrophic chondrocytes cultured in conditions in which they are surrounded by an organized extracellular matrix but do not express transferrin contained only 30% of the chemotactic activity normally observed in the media conditioned by hypertrophic chondrocytes. As expected, this residual activity was not affected by the addition of antibodies directed against the transferrin. From our study it appears that native transferrin in conditioned medium has a higher activity than purified transferrin. A possible synergism between transferrin and other factors present in the medium could explain this finding. An alternative explanation would be the possibility of partial inactivation of transferrin during purification.
In vivo studies on embryonic chorioallantoic membranes showed an angiogenic response to purified transferrins and conditioned culture medium. The angiogenic response of transferrin purified from chondrocyte culture medium and of concentrated conditioned culture medium was comparable to that obtained with bFGF, a well-known angiogenic molecule.
We have shown a role for transferrin in promoting endothelial cell chemotaxis and chemoinvasion in vitro and angiogenesis in an in vivo assay. In evaluating the possible involvement of transferrin in promoting angiogenesis during tissue development, one should consider that the angiogenic process is the result of a balance between angiogenic and antiangiogenic factors. A high expression of transferrin in a given tissue, e.g., the liver, does not necessarily imply implementation of a neoangiogenic program, especially if antiangiogenic or other regulatory factors are also expressed. Transferrin appears to have a unique angiogenic activity, as, unlike other angiogenic factors, it did not promote proliferation in the endothelial cell line we have used for our studies.
Studies to determine the mechanisms of action of transferrin as a chemotactic agent and to identify additional factors acting with transferrin are currently under investigation. It is expected that these studies not only will allow a better understanding of the mechanisms controlling blood vessel invasion in the hypertrophic cartilage during endochondral bone formation, but also will contribute to an understanding of the relationship between control of cell proliferation and angiogenesis in other tissues including metastatic tumors.
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This work was supported by a grant from Progetti Finalizzati "Applicazioni cliniche della ricerca oncologica," Consiglio Nazionale delle Ricerche, Rome, and by funds from the Associazione Italiana per la Ricerca sul Cancro and from Telethon, Italy.
Submitted: 11 April 1996
Revised: 19 December 1996
Address all correspondence to Fiorella Descalzi Cancedda, Centro di Biotecnologie Avanzate, Largo Rosanna Benzi, 10, 16132 Genova, Italy. Tel.: (39) 10-5737392. Fax: (39) 10-5737405. e-mail: Cancedda{at}sirio.cba.unige.it
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