Galectin-3 Induces Endothelial Cell Morphogenesis and Angiogenesis
2000; Elsevier BV; Volume: 156; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64959-0
ISSN1525-2191
AutoresPratima Nangia‐Makker, Yuichiro Honjo, Rebecca Sarvis, Shiro Akahani, Victor Hogan, Kenneth J. Pienta, Avraham Raz,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoIncreasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells. Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells. 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Galectin-3 is a member of a growing family of carbohydrate-binding proteins with molecular masses ranging from 29 to 34 kd. They share an affinity for β-galactoside-containing glycoconjugates and a conserved sequence of the sugar-binding motif (for reviews see refs.32–34). Galectin-3 (also known as Mac-2, CBP-35, ml-34, L-29, hL-31, and εBP. is a Mr ∼30,000 protein composed of two distinct structural motifs, an amino-terminal half containing Gly-X-Y tandem repeats characteristic of collagens and a carboxyl-terminal half containing the carbohydrate-binding site. Galectin-3 is expressed in a wide range of neoplasms and is involved in multiple biological processes through interaction with specific ligands, including cell growth, adhesion, differentiation, inflammation, apoptosis, and metastasis.33Barondes SH Cooper DNW Gitt MA Leffler H Galectins, structure, and function of a large family of animal lectins.J Biol Chem. 1994; 269: 20807-20819Abstract Full Text PDF PubMed Google Scholar, 34Akahani S Nangia-Makker P Inohara H Kim H-RC Raz A Galectin-3, a novel anti-apoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family.Cancer Res. 1997; 57: 5272-5276PubMed Google Scholar In human tumors, a direct relationship between galectin-3 levels and the stage of tumor progression in colon, gastric, thyroid, breast, and head and neck carcinomas has been demonstrated.35Lotan R Ito H Yasui W Yokozaki H Lotan D Tahara E Expression of a 31 kDa lactose-binding lectin in normal human gastric mucosa and in primary and metastatic gastric carcinomas.Int J Cancer. 1994; 56: 474-480Crossref PubMed Scopus (130) Google Scholar, 36Shoeppner HL Raz A Ho SB Bresalier R Expression of an endogenous galactoside-binding lectin correlates with neoplastic progression in the colon.Cancer. 1995; 75: 2818-2826Crossref PubMed Scopus (214) Google Scholar, 37Xu X-C El-Nagger A Lotan R Differential expression of galectin-1 and galectin-3 in thyroid tumors: potential diagnostic implications.Am J Pathol. 1995; 147: 815-822PubMed Google Scholar, 38Nangia-Makker P Thompson EW Hogan C Ochieng J Raz A Induction of tumorigenicity by gal-3 in a non-tumorigenic human breast carcinoma cell line.Int J Oncol. 1995; 7: 1079-1087PubMed Google Scholar, 39Gillenwater A Xu C El-Nagger AK Clayman GN Lotan R Expression of galectins in head and neck squamous cell carcinoma.Head Neck. 1990; 18: 422-432Crossref Google Scholar, 40Fernandez PL Merino MJ Gomez M Campo E Medina T Castronovo V Sanjuan X Liu F-T Sobel M Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue.J Pathol. 1997; 181: 80-86Crossref PubMed Scopus (136) Google Scholar, 41Bresalier RS Mazurek N Sternberg LR Byrd JC Yunker CK Nangia-Makker P Raz A Metastasis of human colon cancer is altered by modifying expression of the β galactoside-binding protein galectin-3.Gastroenterology. 1998; 115: 287-296Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 42Nangia-Makker P Sarvis R Visscher DW Bailey-Penrod J Raz A Sarkar FH Galectin-3 and L1 retrotransposons in human breast carcinomas.Breast Cancer Res Treat. 1998; 49: 171-183Crossref PubMed Scopus (39) Google Scholar It is obvious that the formation of new capillary vessels in a tumor is critical for its continuous growth and provides a gateway for the dissemination of malignant cells. We have previously transfected galectin-3 null human breast carcinoma BT-549 cells with human galectin-3 cDNA and established expressing cell clones that, unlike the parental cells, progressively grew and metastasized in nude mice.38Nangia-Makker P Thompson EW Hogan C Ochieng J Raz A Induction of tumorigenicity by gal-3 in a non-tumorigenic human breast carcinoma cell line.Int J Oncol. 1995; 7: 1079-1087PubMed Google Scholar This raised the question of a possible role for galectin-3 in angiogenesis. We show here that soluble galectin-3 induces endothelial capillary tube formation in vitro and angiogenesis in vivo, and the specificity of the effect is established by antibodies and competitive sugars. In summary, these results suggest that angiogenesis could be mediated by carbohydrate recognition. Human umbilical vein endothelial cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The human breast cancer cell line BT-549 was a gift from Dr. Eric W. Thompson, and its galectin-3-transfected clone (11-9-1-4) was established as previously reported.38Nangia-Makker P Thompson EW Hogan C Ochieng J Raz A Induction of tumorigenicity by gal-3 in a non-tumorigenic human breast carcinoma cell line.Int J Oncol. 1995; 7: 1079-1087PubMed Google Scholar HUV-EC-C were cultured in Ham's F12K medium (Irvine Scientific, Irvine, CA) supplemented with 100 μg/ml heparin (Sigma Chemical Co., St. Louis, MO), 50 μg/ml endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA), and 10% fetal calf serum (FCS) (Summit Biotechnology, Fort Collins, CO). BT-549 and 11-9-1-4 cells were maintained in Dulbecco's minimum essential medium (Gibco, Rockville, MD) containing 10% heat-inactivated FCS, essential and nonessential amino acids (Gibco), vitamins, and antibiotics (Mediatech Inc., Herndon, VA). The cells were maintained in a humidified chamber with 95% air and 5% CO2 at 37°C. The HUV-EC-C were grown to confluence, detached from the monolayer with 0.25% trypsin, 2 mmol/L EDTA, and split 1:2. the breast cancer cell lines were split 1:4 for further cultivation. For collection of the conditioned media, 4 × 106 human breast carcinoma cells were plated on a 60-mm tissue culture dish. The next day, the medium was replaced with serum-free medium after thorough washing with phosphate-buffered saline (PBS). The medium was collected 3 days later, concentrated 10-fold by centrifugation through ultrafree MC filter units (Millipore, Bedford, MA) with a 10,000-kd cut point, and analyzed for the presence of galectin-3 by Western blotting, using anti galectin-3 mAb. Human recombinant galectin-3 was isolated and affinity purified as described.43Ochieng J Platt D Tait L Hogan V Raz T Carmi P Raz A Structure function relationship of a recombinant human galactoside-binding protein.Biochemistry. 1993; 32: 4455-4460Crossref PubMed Scopus (95) Google Scholar Briefly, galectin-3 cDNA was expressed in Escherichia coli HMS174, using pET5a expression vector (Promega, Madison, WI). The protein was purified from the bacterial cell lysates, using an asialofetuin affinity column followed by extensive dialysis against PBS (pH 7.4). The pAb was prepared in rabbits against purified human recombinant galectin-3 (Genemed Biotechnologies, S. San Francisco, CA). The anti-galectin-3 mAb-producing hybridoma TIB-166 was purchased from ATCC. Mouse anti-β3-integrin monoclonal antibody was purchased from Transduction Laboratories (San Diego, CA). Anti-von Willebrand factor polyclonal antibody raised in rabbit was obtained from Sigma Chemical Co. Citrus pectin was purchased from Sigma Chemical Co. and modified into modified citrus pectin (MCP) as already described.44Platt D Raz A Modulation of the lung colonization of B16–F1 melanoma cells by citrus pectin.J Natl Cancer Inst. 1992; 84: 438-442Crossref PubMed Scopus (192) Google Scholar To prepare Matrigel,45Bauer J Margolism M Schreiner C Edgell C-J Azizkhan J Lazarowski E Juliano RL In vitro model of angiogenesis using a human endothelium derived permanent cell line contributions of induced gene expression, G proteins, and integrins.J Cell Physiol. 1992; 153: 437-449Crossref PubMed Scopus (171) Google Scholar 200 μl of Matrigel (Collaborative Biomedical Products, MA) thawed on ice was added to each chamber of an eight-chamber slide. The air bubbles were carefully removed, and the slide was transferred to a 37°C incubator for 15 minutes. After gelation, 5 × 104 endothelial cells, which were separated from monolayers with trypsin treatment, were plated onto the gel in 200 μl medium. In some chambers, specific antibodies or PIS was added to the cells at the time of plating on Matrigel. The slides were incubated for 16 hours at 37°C. Alternatively, the Matrigel was diluted to 4.5 mg/ml with serum-free F12K medium and allowed to gel for 2 hours at 37°C. A total of 2 × 104 cells/200 μl medium were plated per chamber. Purified recombinant or secreted galectin-3 was added to some chambers. In some experiments galectin-3 was added with or without a competitive disaccharide (lactose), a competitive polysaccharide (MCP), and a noncompetitive disaccharide (sucrose), and endothelial cell migration and rearrangement was visualized after 4–6 hours. Endothelial cells were incubated with 10 μg/ml galectin-3 and lysed at different time intervals. The cell lysates or, in some experiments, conditioned media were suspended in reducing Tris-sodium dodecyl sulfate sample buffer, boiled for 5 minutes, and separated on a 8% or 12.5% polyacrylamide separating gel and 3.5. stacking gel. The separated proteins were then electroblotted to polyvinyl pyrrolidine fluoride (PVDF) membranes (MSI, Westborough, MA) and quenched in 5% nonfat dried milk in PBS for 4 hours, followed by incubation with the first antibody for 1 hour at room temperature. Subsequently, the membrane was washed five times with quench solution containing 0.1% Tween-20 and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG or rabbit anti-rat IgG + IgM; Zymed, S. San Francisco, CA) for 1 hour and washed as above and processed for chemiluminescence. To determine the binding affinity and number of receptors on the endothelial cell surface, galectin-3 was iodinated in the presence of chloramine T. Briefly, 1 μg galectin-3 was incubated with 250 μCi Na125I in the presence of 40 μg chloramine T and 100 μl H2O on ice for 1 minute. The reaction was stopped by the addition of 20 μl of 1 mol/L KI. To remove the unbound labeled iodine, the reaction mixture was spun through Ultrafree-CL centrifugal filters (Millipore) precoated with 0.1% bovine serum albumin (BSA) in PBS. The labeled galectin-3 was resuspended in 50 μl of 0.1% BSA in 1× PBS and stored at 4°C for a maximum period of 1 month. One day before the assay HUV-EC-C were plated at a density of 5 × 104 cells per well in a 24-chamber plate. After washing, the wells were blocked with 0.1% BSA in 1× PBS for 30 minutes and incubated with 1–6 ng iodinated galectin-3/well in the presence or absence of 50 mmol/L lactose. After 2 hours of incubation at 4°C with constant shaking, the solution was removed and the cells were washed three times with 0.1% BSA-PBS. To measure the bound galectin-3, the cells were lysed with 1 mol/L NaOH for 30 minutes at room temperature, and the radioactivity was measured with a gamma counter. For Scatchard plot analysis, the cells were incubated with 0.5 ng of iodinated galectin-3 in 0.1% BSA and 100 μl of 0.1% BSA containing a serial dilution of cold galectin-3 ranging from 0 to 50 ng for 2 hours at 4°C with constant shaking at 60 cycles/minute. The washes and counting were done as above. Values of ligand-receptor and [ligand receptor]/free ligand were calculated as follows: Ligand receptor: [cold galectin-3(ng) + iodinated galectin-3 (ng)] × [bound galectin-3 (cpm)]/[Total counts added (cpm)] Ligand receptor/free ligand: bound galectin-3 (cpm)/total cpm − bound cpm The affinity of the receptor (KD value) and receptor concentration (R value) were determined using the method of Scatchard.46Scatchard G The attractions of proteins for small molecules and ions.Ann NY Acad Sci. 1949; 51: 660-672Crossref Scopus (17815) Google Scholar The assay was performed with a Boyden chamber. Briefly, 30 μl of conditioned medium from the galectin-3-secreting cells (11-9-1-4) and null galectin-3 cells (BT-549) and recombinant galectin-3 (0–20 μg/ml) in serum-free F12K medium were added to the lower chamber as a chemoattractant. HUV-EC-C (5 × 104) were added to the upper chamber. The two chambers were separated with polycarbonate filters (8-μm pore size) and incubated at 37°C for 5 hours. At the end of the incubation period, the cells attached to the lower surface of the filter were fixed and stained using the Diff-Quik Stain set (Baxter, IL). A total of 10 fields were counted from each chamber under a microscope, and their sum was plotted. Each assay was carried out in quadruplicate. Galectin-3-expressing (11-9-1-4) or nonexpressing (BT-549. cells38Nangia-Makker P Thompson EW Hogan C Ochieng J Raz A Induction of tumorigenicity by gal-3 in a non-tumorigenic human breast carcinoma cell line.Int J Oncol. 1995; 7: 1079-1087PubMed Google Scholar were injected subcutaneously into the dorsolateral region of nude mice (five per group) in the presence of Matrigel (1 × 106 cells in 300 μl per mouse). After 10 days the tumors were removed along with the overlying skin, fixed with 10% buffered formalin, and processed for embedding, sectioning, and immunohistochemistry as described below. NCR nude mice were also injected subcutaneously dorsolaterally with 0.4 ml Matrigel alone or in combination with 5 μg/ml bFGF or 10 μg/ml galectin-3 according to the method of Passaniti et al.47Passaniti A Taylor RM Pili R Guo Y Long PV Haney JA Pauly RR Grant DS Martin GR A simple quantitative method for assessing angiogenesis and anti-angiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor.Lab Invest. 1992; 67: 519-528PubMed Google Scholar The injected Matrigel rapidly formed a solid gel that persisted for at least 10 days in mice. The mice were euthanized after 6 days, and the mass of Matrigel was removed along with overlying skin and fixed with 10% formaldehyde for at least 24 hours before it was embedded in paraffin. The paraffin blocks were then cut into 4-μm-thick sections and processed for immunohistochemistry. Immunohistochemistry was performed using a modification of the avidin-biotin peroxidase complex technique. Briefly, 4-μm tissue sections were deparaffinized, rehydrated, and placed in 3% hydrogen peroxide to inhibit endogenous peroxidase. The tissue sections were trypsinized with 0.1% trypsin and 0.1% CaCl2 for 30 minutes at 37°C to expose the antigenic sites masked by formalin fixation, blocked for 1 hour with 3% normal goat serum (Sigma Chemical Co.), and subsequently incubated with antibody against von Willebrand factor for 60 minutes at a dilution of 1:1000. The sections were then treated with biotinylated secondary antibody (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, followed by avidin biotin complex reagent for 30 minutes and diaminobenzidine (Sigma Chemical Co) for 1 minute. Counterstaining was performed with hematoxylin. For galectin-3 staining, basically a similar protocol was followed with a few modifications. The sections were not trypsinized, as formaldehyde fixation did not mask the antigenic sites of galectin-3. Blocking was performed with 3% normal rabbit serum. The primary antibody used was monoclonal antibody produced by TIB-166 hybridoma at a dilution of 1:2 for 60 minutes. To establish the tube-forming property of endothelial cells on Matrigel, HUV-EC-C were plated on a gel formed by 200 μl of 14 mg/ml or 4.5 mg/ml Matrigel. On a gel prepared with undiluted Matrigel there was a rapid organization of endothelial cells into capillary-like structures (Figure 1A), whereas on diluted Matrigel, the cells underwent migration and organization into elongated structures without an interconnecting network (Figure 2A).Figure 2Capillary tube formation on Matrigel. A: Control; B: polyclonal antibody; C: preimmune serum. Scale bar, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To study the effect of galectin-3 on endothelial cell tube formation, we have analyzed the dose response of HUV-EC-C to soluble human galectin-3. Cells (5 × 104) were plated on a gel formed by diluted Matrigel, in the presence of varying concentrations of galectin-3. Figure 1, A–D, show a concentration-dependent anastomosing network of endothelial cells. Similar stimulation of organization into a network was observed when HUV-EC-C were incubated with conditioned medium collected from the transfected human breast carcinoma 11-9-1-4 cells (Figure 1E), which secrete galectin-3 (inset: Lane 1). The conditioned medium collected from the galectin-3 null parental BT-549 cell culture (inset: Lane 2. failed to enhance the organization (Figure 1F), suggesting that this stimulation is not only restricted to the recombinant form but could also be mediated by cellular processed galectin-3. To further verify the importance of galectin-3, HUV-EC-C were plated on undiluted Matrigel in the presence and absence of a specific antibody or the preimmune serum. Figure 2, A–C, demonstrates that the basement-membrane-induced HUV-EC-C differentiation could be inhibited by anti-galectin-3 polyclonal antibody and not by preimmune serum control. There was a 50–90% reduction in the number of capillaries formed in the presence of pAb in various experiments. However, the mAb against the amino-terminal domain failed to inhibit tube formation (not shown). Next, we questioned whether the effect of galectin-3 is mediated by its carbohydrate-binding domain; thus the effect of competitive sugars
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