Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Abstract

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis. Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis. basic fibroblast growth factor endothelial basal medium epidermal growth factor receptor endothelial growth medium high-performance thin-layer chromatography human umbilical vein endothelial cell 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N-butyldeoxynojirimycin vascular endothelial growth factor vascular endothelial growth factor receptor 2 Gangliosides are a family of sialic acid-containing glycosphingolipids that are enriched in the outer surface of plasma membranes and have long been associated with tumor malignancy and metastasis (1.Alessandri G. Filippeschi S. Sinibaldi P. Mornet F. Passera P. Spreafico F. Cappa P.M. Gullino P.M. Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.Cancer Res. 1987; 47: 4243-4247PubMed Google Scholar, 2.Bai H. Seyfried T.N. Influence of ganglioside GM3 and high density lipoprotein (HDL) on the cohesion of mouse brain tumor cells.J. Lipid Res. 1997; 38: 160-172Abstract Full Text PDF PubMed Google Scholar, 3.Hakomori S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism.Cancer Res. 1996; 56: 5309-5318PubMed Google Scholar). These molecules contain an oligosaccharide head group that is attached to a lipophilic ceramide, consisting of a sphingosine base and a long-chain fatty acid. Gangliosides can be shed from the surface of tumor cells into the microenvironment, where they can influence tumor host cell interactions to include angiogenesis (1.Alessandri G. Filippeschi S. Sinibaldi P. Mornet F. Passera P. Spreafico F. Cappa P.M. Gullino P.M. Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.Cancer Res. 1987; 47: 4243-4247PubMed Google Scholar, 4.Zeng G. Gao L. Birkle S. Yu R.K. 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Ganglioside GM3, a simple monosialoganglioside (NeuAca2→3Galb1→4Glcb1→1′-ceramide), modulates cell adhesion, proliferation, and differentiation (2.Bai H. Seyfried T.N. Influence of ganglioside GM3 and high density lipoprotein (HDL) on the cohesion of mouse brain tumor cells.J. Lipid Res. 1997; 38: 160-172Abstract Full Text PDF PubMed Google Scholar, 5.Alessandri G. Ferraris P.Cornaglia Gullino P.M. Angiogenic and angiostatic microenvironment in tumors—role of gangliosides.Acta Oncol. 1997; 36: 383-387Crossref PubMed Scopus (35) Google Scholar, 12.Manfredi M.G. Lim S. Claffey K.P. Seyfried T.N. Gangliosides influence angiogenesis in an experimental mouse brain tumor.Cancer Res. 1999; 59: 5392-5397PubMed Google Scholar, 14.Mitsuzuka K. Handa K. Satoh M. Arai Y. Hakomori S. A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.J. Biol. 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In contrast to GM3, complex gangliosides like GM2, GM1, GD1a, GD1b, GT1b, and GD3, which contain longer oligosaccharide chains than that of GM3, enhance tumor cell proliferation, invasion, and metastasis (1.Alessandri G. Filippeschi S. Sinibaldi P. Mornet F. Passera P. Spreafico F. Cappa P.M. Gullino P.M. Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.Cancer Res. 1987; 47: 4243-4247PubMed Google Scholar, 3.Hakomori S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism.Cancer Res. 1996; 56: 5309-5318PubMed Google Scholar, 14.Mitsuzuka K. Handa K. Satoh M. Arai Y. Hakomori S. A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.J. Biol. Chem. 2005; 280: 35545-35553Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18.De Cristan G. Morbidelli L. Alessandri G. Ziche M. Cappa A.P. Gullino P.M. Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium.J. Cell. Physiol. 1990; 144: 505-510Crossref PubMed Scopus (34) Google Scholar, 19.Ravindranath M.H. Tsuchida T. Morton D.L. Irie R.F. Ganglioside GM3:GD3 ratio as an index for the management of melanoma.Cancer. 1991; 67: 3029-3035Crossref PubMed Scopus (93) Google Scholar). Increased tumorigenic effects of complex gangliosides were observed in a variety of tumor cells, including bladder, lymphoma, glioma, neuroblastoma, and melanoma (7.Valentino L.A. Ladisch S. Localization of shed human tumor gangliosides: association with serum lipoproteins.Cancer Res. 1992; 52: 810-814PubMed Google Scholar, 11.Ladisch S. Kitada S. Hays E.F. Gangliosides shed by tumor cells enhance tumor formation in mice.J. Clin. Invest. 1987; 79: 1879-1882Crossref PubMed Scopus (123) Google Scholar, 14.Mitsuzuka K. Handa K. Satoh M. Arai Y. Hakomori S. A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.J. Biol. Chem. 2005; 280: 35545-35553Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 20.Shochat S.J. Corbelletta N.L. Repman M.A. Schengrund C.L. A biochemical analysis of thoracic neuroblastomas: a Pediatric Oncology Group study.J. Pediatr. Surg. 1987; 22: 660-664Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 21.Chu S.H. Ma Y.B. Zhang H. Feng D.F. Zhu Z.A. Li Z.Q. Yuan X.H. Hepatocyte growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells.J. Neurooncol. 2007; 85: 33-38Crossref PubMed Scopus (11) Google Scholar, 22.Koochekpour S. Merzak A. Pilkington G.J. Vascular endothelial growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells in vitro.Cancer Lett. 1996; 102: 209-215Crossref PubMed Scopus (109) Google Scholar). Specific inhibitors of ganglioside biosynthesis also reduced tumor growth (23.Ranes M.K. El-Abbadi M. Manfredi M.G. Mukherjee P. Platt F.M. Seyfried T.N. N-Butyldeoxynojirimycin reduces growth and ganglioside content of experimental mouse brain tumours.Br. J. Cancer. 2001; 84: 1107-1114Crossref PubMed Scopus (40) Google Scholar, 24.Weiss M. Hettmer S. Smith P. Ladisch S. Inhibition of melanoma tumor growth by a novel inhibitor of glucosylceramide synthase.Cancer Res. 2003; 63: 3654-3658PubMed Google Scholar, 25.Deng W. Li R. Ladisch S. Influence of cellular ganglioside depletion on tumor formation.J. Natl. Cancer Inst. 2000; 92: 912-917Crossref PubMed Scopus (87) Google Scholar), whereas gene-linked shifts in ganglioside distribution changed tumor growth and angiogenesis in vivo (4.Zeng G. Gao L. Birkle S. Yu R.K. Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production.Cancer Res. 2000; 60: 6670-6676PubMed Google Scholar, 8.Abate L.E. Mukherjee P. Seyfried T.N. Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma.J. Neurochem. 2006; 98: 1973-1984Crossref PubMed Scopus (24) Google Scholar, 12.Manfredi M.G. Lim S. Claffey K.P. Seyfried T.N. Gangliosides influence angiogenesis in an experimental mouse brain tumor.Cancer Res. 1999; 59: 5392-5397PubMed Google Scholar).Endothelial cell signaling is important in cancer-associated vascularity (angiogenesis). The proliferation and migration of endothelial cells in response to growth factors is one of the major determinants of tumor growth and progression. Dysregulation of the balance between proangiogenic and antiangiogenic factors contributes to the abnormal vasculature in tumors. The targeting of tumor endothelial cells, therefore, is considered important for managing tumor growth (1.Alessandri G. Filippeschi S. Sinibaldi P. Mornet F. Passera P. Spreafico F. Cappa P.M. Gullino P.M. Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.Cancer Res. 1987; 47: 4243-4247PubMed Google Scholar, 8.Abate L.E. Mukherjee P. Seyfried T.N. Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma.J. Neurochem. 2006; 98: 1973-1984Crossref PubMed Scopus (24) Google Scholar, 26.Folkman J. The role of angiogenesis in tumor growth.Semin. Cancer Biol. 1992; 3: 65-71PubMed Google Scholar).Vascular endothelial cells are responsive to a number of proangiogenic growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which promote endothelial cell survival, growth, and migration (18.De Cristan G. Morbidelli L. Alessandri G. Ziche M. Cappa A.P. Gullino P.M. Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium.J. Cell. Physiol. 1990; 144: 505-510Crossref PubMed Scopus (34) Google Scholar, 27.Claffey K.P. Abrams K. Shih S.C. Brown L.F. Mullen A. Keough M. Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.Lab. Invest. 2001; 81: 61-75Crossref PubMed Scopus (86) Google Scholar, 28.Ferrara N. Smyth T.Davis The biology of vascular endothelial growth factor.Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (4014) Google Scholar). Interestingly, complex gangliosides enhance the response of endothelial cells to the proangiogenic action of bFGF and VEGF (4.Zeng G. Gao L. Birkle S. Yu R.K. Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production.Cancer Res. 2000; 60: 6670-6676PubMed Google Scholar, 13.Liu Y. McCarthy J. Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling.Cancer Res. 2006; 66: 10408-10414Crossref PubMed Scopus (37) Google Scholar, 18.De Cristan G. Morbidelli L. Alessandri G. Ziche M. Cappa A.P. Gullino P.M. Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium.J. Cell. Physiol. 1990; 144: 505-510Crossref PubMed Scopus (34) Google Scholar, 29.Lang Z. Guerrera M. Li R. Ladisch S. Ganglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration.Biochem. Biophys. Res. Commun. 2001; 282: 1031-1037Crossref PubMed Scopus (55) Google Scholar). GD1a enrichment of endothelial cells enhanced VEGF receptor dimerization, autophosphorylation, and downstream signaling pathways for endothelial cell proliferation and migration (13.Liu Y. McCarthy J. Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling.Cancer Res. 2006; 66: 10408-10414Crossref PubMed Scopus (37) Google Scholar). The involvement of gangliosides in angiogenesis is dependent on the intact molecules, as neither asialo species nor sialic acid alone influences angiogenesis (30.Ziche M. Alessandri G. Gullino P.M. Gangliosides promote the angiogenic response.Lab. Invest. 1989; 61: 629-634PubMed Google Scholar). In contrast to the enhancing effects of complex gangliosides on angiogenesis, GM3 reduces endothelial cell proliferation and migration (5.Alessandri G. Ferraris P.Cornaglia Gullino P.M. Angiogenic and angiostatic microenvironment in tumors—role of gangliosides.Acta Oncol. 1997; 36: 383-387Crossref PubMed Scopus (35) Google Scholar, 8.Abate L.E. Mukherjee P. Seyfried T.N. Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma.J. Neurochem. 2006; 98: 1973-1984Crossref PubMed Scopus (24) Google Scholar, 12.Manfredi M.G. Lim S. Claffey K.P. Seyfried T.N. Gangliosides influence angiogenesis in an experimental mouse brain tumor.Cancer Res. 1999; 59: 5392-5397PubMed Google Scholar, 31.Ziche M. Morbidelli L. Alessandri G. Gullino P.M. Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio.Lab. Invest. 1992; 67: 711-715PubMed Google Scholar). Little is known, however, about the molecular mechanism by which GM3 inhibits angiogenesis.The interactions of gangliosides with cell surface receptor molecules in tumor cells as well as in endothelial cells may be critical for the tumor-induced progression of the microenvironment (8.Abate L.E. Mukherjee P. Seyfried T.N. Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma.J. Neurochem. 2006; 98: 1973-1984Crossref PubMed Scopus (24) Google Scholar, 13.Liu Y. McCarthy J. Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling.Cancer Res. 2006; 66: 10408-10414Crossref PubMed Scopus (37) Google Scholar, 14.Mitsuzuka K. Handa K. Satoh M. Arai Y. Hakomori S. A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.J. Biol. Chem. 2005; 280: 35545-35553Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The inhibitory effects of GM3 on the epidermal growth factor receptor (EGFR) tyrosine kinase are well studied (32.Wang X.Q. Sun P. Paller A.S. Ganglioside GM3 blocks the activation of epidermal growth factor receptor induced by integrin at specific tyrosine sites.J. Biol. Chem. 2003; 278: 48770-48778Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 33.Bremer E.G. Schlessinger J. Hakomori S. Ganglioside-mediated modulation of cell growth.J. Biol. Chem. 1986; 261: 2434-2440Abstract Full Text PDF PubMed Google Scholar, 34.Yoon S.J. Nakayama K. Hikita T. Handa K. Hakomori S.I. Epidermal growth factor receptor tyrosine kinase is modulated by GM3 interaction with N-linked GlcNAc termini of the receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 18987-18991Crossref PubMed Scopus (164) Google Scholar). Yoon et al. (34.Yoon S.J. Nakayama K. Hikita T. Handa K. Hakomori S.I. Epidermal growth factor receptor tyrosine kinase is modulated by GM3 interaction with N-linked GlcNAc termini of the receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 18987-18991Crossref PubMed Scopus (164) Google Scholar) showed that GM3 inhibits the EGFR tyrosine kinase through interactions with N-acetylglucosamine residues on the glycan units of the receptor. In contrast to the information available on the influence of GM3 on the EGFR, little is known about the influence of GM3 on other growth factor receptors, including the vascular endothelial growth factor receptor (VEGFR). VEGFR-2 or KDR is phosphorylated upon VEGF stimulation, which induces the phosphoinositide-3 kinase/Akt pathway, resulting in enhanced endothelial proliferation and migration (35.Veikkola T. Karkkainen M. Claesson-Welsh L. Alitalo K. Regulation of angiogenesis via vascular endothelial growth factor receptors.Cancer Res. 2000; 60: 203-212PubMed Google Scholar, 36.Gille H. Kowalski J. Li B. LeCouter J. Moffat B. Zioncheck T.F. Pelletier N. Ferrara N. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.J. Biol. Chem. 2001; 276: 3222-3230Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar).In this study, we examined the influence of the exogenous addition of GM3 on the proliferation of human umbilical vein endothelial cells (HUVECs) in the presence of VEGF and the VEGF enhancer, GD1a. We show that GM3 suppresses angiogenesis both in vitro and in vivo. We also found that GM3 enrichment of HUVECs inhibits migration toward VEGF as a chemoattractant. Additionally, we found that GM3 reduced VEGFR-2 phosphorylation and downstream Akt signaling in HUVECs, suggesting a mechanism by which GM3 reduces endothelial cell proliferation and migration. Moreover, pharmacological depletion of endogenous GM3 significantly increased HUVEC proliferation and VEGFR-2 and Akt phosphorylation.MATERIALS AND METHODSReagentsPurified disialoganglioside GD1a (bovine brain), recombinant human vascular endothelial growth factor (VEGF165), and BSA were purchased from Sigma (St. Louis, MO). Highly purified monosialoganglioside GM3 was purchased from Matreya (Pleasant Gap, PA). The commercial ganglioside preparations were the same as those used by other groups (13.Liu Y. McCarthy J. Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling.Cancer Res. 2006; 66: 10408-10414Crossref PubMed Scopus (37) Google Scholar, 14.Mitsuzuka K. Handa K. Satoh M. Arai Y. Hakomori S. A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9.J. Biol. Chem. 2005; 280: 35545-35553Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) but were not checked for purity. Matrigel™ basement membrane matrix was purchased from BD Biosciences (Bedford, MA). The CellTiter 96 nonradioactive cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit was from Promega (Madison, WI). Endothelial growth medium (EGM-2), BulletKit medium, and supplement-free endothelial basal medium (EBM) were purchased from Cambrex (Charles City, IA). Ganglioside GD1a and VEGF were dissolved in EBM and sonicated, and aliquots stored at −20°C. Ganglioside GM3 was prepared in the same manner but was first dissolved in chloroform-ethanol (1:1, v/v) and was then evaporated and dissolved in EBM. Antibodies against total and phosphorylated VEGFR-2 and Akt were purchased from Cell Signaling (Beverly, MA). FITC-labeled Isolectin B4 was purchased from Vector Laboratories (Burlingame, CA). Calcein was purchased from Invitrogen (San Diego, CA). Anti-GM3 DH2 antibody was a gift from Dr. S. I. Hakomori (University of Washington, Seattle). Cy5-labeled anti-mouse antibody was purchased from Jackson ImmunoResearch (West Grove, PA). N-butyldeoxynojirimycin (NB-DNJ; molecular weight, 219.3) was obtained as a gift from Oxford Glycosciences (Abigdon, UK). [14C]galactose was obtained from Sigma.Cell cultureHUVECs (pooled) were purchased from Cambrex and were maintained at 37°C in EGM-2 in humidified air containing 5% CO2. All experiments with HUVECs were conducted at passages two to six.AnimalsBALBc/J-SCID mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were used for the Matrigel plug study. All animal experiments were carried out with ethical committee approval in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the institutional care committee.Proliferation assayIn vitro HUVEC proliferation was analyzed using the CellTiter 96 nonradioactive cell proliferation assay according to the manufacturer's protocol. Briefly, 5 × 103 HUVECs in EGM-2 were seeded in each well of a 96-well plate. After 24 h, the HUVECs were washed and treated with 20 μM GD1a and GM3 in EBM for 24 h. After this incubation, the ganglioside-containing medium was removed. The HUVECs were then washed with EBM and were stimulated with 4 ng/ml VEGF in EBM for 24 h. For the NB-DNJ study, cells were treated with 200 μM NB-DNJ in the absence and presence of GM3 for 48 h and were then stimulated with 4 ng/ml VEGF in EBM for an additional 24 h. Tetrazolium salt dye solution (15 μl) was added to each well, and the plate was returned to the incubator for 4 h. Stop solution (100 μl) was then added to solubilize the metabolite and to lyse the HUVECs. The plate was incubated for 24 h. The plate was shaken for 30 s after incubation and was read in a microplate reader (SpectraMax M5; Molecular Devices) at 595 nm. This procedure was performed in triplicate.Flow cytometryHUVECs were treated with 0, 0.5, 2.5, or 5.0 μM GM3 in EBM for 24 h at 37°C in 5% CO2-humidified conditions. Confluent cells were detached with trypsin/EDTA and were washed with PBS. HUVECs (2 × 105) were incubated with anti-GM3 DH2 antibody (1:50) for 1 h on ice, washed with PBS, and incubated with 1:500 Cy5 anti-mouse secondary antibody for 30 min on ice in darkness. Cells were then washed twice with PBS, resuspended, and analyzed with FACSDiva software (Beckman Coulter). Control experiments included HUVECs treated with only secondary antibody.Migration assayConfluent HUVECs in T75 flasks were rinsed and treated with 20 μM GM3 in EBM containing 0.1% BSA for a minimum of 4–5 h at 37°C in 5% CO2-humidified conditions. A control flask of HUVECs was treated with EBM containing 0.1% BSA. Cells were washed twice in EBM and were harvested in EBM containing 0.1% BSA. According to the manufacturer's protocol, equal numbers of control and treated cells (4 × 105 cells/ml) in 250 μl of EBM were seeded on the upper insert of a 24-well migration chamber (BD Falcon Fluoroblok Insert system; 3.0 μm pore size). EBM (750 μl) containing 100 ng/ml VEGF was then placed in the lower chamber according to the protocol. This VEGF concentration was recommended and necessary for an effective migration of HUVECs. Serum-containing medium was used as a positive control for these experiments. The plates were incubated for 24 h under the humidified conditions as above. HUVECs that migrated to the underside of the membrane were washed with HBSS, and the inserts were transferred in the companion wells containing calcein (4–5 μg/ml) solution. HUVECs were incubated in calcein for 1 h and washed, and the fluorescently labeled cells were photographed using a fluorescence microscope. Fluorescence was measured using a SpectraMax M5 microplate reader (Molecular Devices).In vivo Matrigel plug assayAngiogenesis was analyzed using the in vivo Matrigel plug assay as we described previously (12.Manfredi M.G. Lim S. Claffey K.P. Seyfried T.N. Gangliosides influence angiogenesis in an experimental mouse brain tumor.Cancer Res. 1999; 59: 5392-5397PubMed Google Scholar, 37.Mukherjee P. El-Abbadi M.M. Kasperzyk J.L. Ranes M.K. Seyfried T.N. Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model.Br. J. Cancer. 2002; 86: 1615-1621Crossref PubMed Scopus (129) Google Scholar). Briefly, Matrigel (200 μl) and EBM (100 μl) containing GD1a and/or GM3 (at 20 or 40 μM) were thoroughly mixed at 4°C. Control plugs contained only the Matrigel/EBM solution. Male BALBc/SCID mice were anesthetized with Avertin (0.1 ml/10 g body weight) and then injected with Matrigel with or without gangliosides subcutaneously in the dorsal midline using a prechilled tuberculin syringe (27 gauge needle). Seven days after implantation and 30 min before necropsy, mice were injected intravenously with 100 μl of FITC-conjugated Griffonia simplicifolia Isolectin B4 (0.25 mg/ml) to stain vascular endothelial cells (38.Wakasugi K. Slike B.M. Hood J. Otani A. Ewalt K.L. Friedlander M. Cheresh D.A. Schimmel P. A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.Proc. Natl. Acad. Sci. USA. 2002; 99: 173-177Crossref PubMed Scopus (231) Google Scholar). Matrigel plugs with the surrounding skin were removed as described previously, and vascularity was photographed (37.Mukherjee P. El-Abbadi M.M. Kasperzyk J.L. Ranes M.K. Seyfried T.N. Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model.Br. J. Cancer. 2002; 86: 1615-1621Crossref PubMed Scopus (129) Google Scholar). Lectin-FITC was extracted from plugs by homogenizing in 500 μl of radioimmunoprecipitation buffer. The homogenate was centrifuged at 1,000 g, and fluorescence was measured at 490 nm using a SpectraMax M5 microplate reader as above.Ganglioside biosynthesis in HUVECsSynthesized gangliosides were isolated from control and NB-DNJ-treated HUVECs as described previously (2.Bai H. Seyfried T.N. Influence of ganglioside GM3 and high density lipoprotein (HDL) on the cohesion of mouse brain tumor cells.J. Lipid Res. 1997; 38: 160-172Abstract Full Text PDF PubMed Google Scholar). Briefly, HUVECs were grown for 72 h in EGM-2 containing 5 μCi of [14C]galactose and 200 μM NB-DNJ. The radiolabeled cells were removed from the flask with a cell scraper in PBS and were collected as a pellet. Before ganglioside isolation, unlabeled mouse ependymoblastoma gangliosides, containing GM3, were added as unlabeled carrier (2.Bai H. Seyfried T.N. Influence of ganglioside GM3 and high density lipoprotein (HDL) on the cohesion of mouse brain tumor cells.J. Lipid Res. 1997; 38: 16