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The Galectin Profile of the Endothelium

2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês

10.2353/ajpath.2008.070938

1525-2191

Victor L. Thijssen, Sarah Hulsmans, Arjan W. Griffioen,

Signaling Pathways in Disease

We previously identified overexpression of galectin-1 in activated tumor endothelium. Currently, the tumor vasculature is a target for therapeutic approaches. Little is known about galectin expression and regulation in the tumor vasculature. Here, we report the expression of galectin-1/-3/-8/-9 in the endothelium as determined by quantitative PCR, Western blot, flow cytometry, and immunohistochemistry. Galectin-2/-4/-12 were detectable at the mRNA level, albeit very low. Galectin-8 and -9 displayed alternative splicing. Immunohistochemistry of normal tissues revealed a broad but low expression of galectin-1 in the vasculature, whereas the expression levels and localization of the other galectins varied. Endothelial cell activation in vitro significantly increased the expression of galectin-1 (5.32 ± 1.97; P = 0.04) and decreased the expression of both galectin-8 (0.59 ± 0.12; P < 0.04) and galectin-9 (0.32 ± 0.06; P < 0.002). Galectin-3 expression was unaltered. Although a portion of these proteins is expressed intracellularly, the membrane protein level of galectin-1/-8/-9 was significantly increased on cell activation in vitro, 6-fold (P = 0.005), 3-fold (P = 0.002), and 1.4-fold (P = 0.04), respectively. Altered expression levels and cellular localization was also observed in vivo in the endothelium of human tumor tissue compared with normal tissue. These data show that endothelial cells express several members of the galectin family and that their expression and distribution changes on cell activation, resulting in a different profile in the tumor vasculature. This offers opportunities to develop therapeutic strategies that are independent of tumor type. We previously identified overexpression of galectin-1 in activated tumor endothelium. Currently, the tumor vasculature is a target for therapeutic approaches. Little is known about galectin expression and regulation in the tumor vasculature. Here, we report the expression of galectin-1/-3/-8/-9 in the endothelium as determined by quantitative PCR, Western blot, flow cytometry, and immunohistochemistry. Galectin-2/-4/-12 were detectable at the mRNA level, albeit very low. Galectin-8 and -9 displayed alternative splicing. Immunohistochemistry of normal tissues revealed a broad but low expression of galectin-1 in the vasculature, whereas the expression levels and localization of the other galectins varied. Endothelial cell activation in vitro significantly increased the expression of galectin-1 (5.32 ± 1.97; P = 0.04) and decreased the expression of both galectin-8 (0.59 ± 0.12; P < 0.04) and galectin-9 (0.32 ± 0.06; P < 0.002). Galectin-3 expression was unaltered. Although a portion of these proteins is expressed intracellularly, the membrane protein level of galectin-1/-8/-9 was significantly increased on cell activation in vitro, 6-fold (P = 0.005), 3-fold (P = 0.002), and 1.4-fold (P = 0.04), respectively. Altered expression levels and cellular localization was also observed in vivo in the endothelium of human tumor tissue compared with normal tissue. These data show that endothelial cells express several members of the galectin family and that their expression and distribution changes on cell activation, resulting in a different profile in the tumor vasculature. This offers opportunities to develop therapeutic strategies that are independent of tumor type. Galectins are a family of proteins that share a binding affinity for β-galactoside-containing carbohydrates. Several members of this family are emerging as targets for cancer therapy. Apart from a direct role in cell transformation, their main contribution to tumor progression involves modification of the antitumor immune response and enhancement of the metastatic potential of tumor cells.1Liu FT Rabinovich GA Galectins as modulators of tumour progression.Nat Rev Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1219) Google Scholar There are several reports showing altered galectin expression profiles in tumor cells of different origin,2van den Brûle F Califice S Castronovo V Expression of galectins in cancer: a critical review.Glycoconj J. 2004; 19: 537-542Crossref PubMed Scopus (171) Google Scholar, 3Huflejt ME Leffler H Galectin-4 in normal tissues and cancer.Glycoconj J. 2004; 20: 247-255Crossref PubMed Scopus (145) Google Scholar, 4Bidon-Wagner N Le Pennec JP Human galectin-8 isoforms and cancer.Glycoconj J. 2004; 19: 557-563Crossref PubMed Scopus (100) Google Scholar and compounds that interfere with galectin function in tumor cells are therefore considered for cancer therapy. An attractive site for therapeutic applications is the endothelium, ie, the monolayer of endothelial cells lining blood vessels. Endothelial cells in the tumor vasculature are easily accessible and less prone to become drug resistant, and disrupting the tumor endothelium results in massive death of tumor cells.5Griffioen AW Molema G Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.Pharmacol Rev. 2000; 52: 237-268PubMed Google Scholar Moreover, tumor endothelial cells express molecules that allow specific targeting independent of the tumor type eg, integrin αVβ3, CD44v3, and CD105.6Brooks PC Montgomery AM Rosenfeld M Reisfeld RA Hu T Klier G Cheresh DA Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2193) Google Scholar, 7Thorpe PE Burrows FJ Antibody-directed targeting of the vasculature of solid tumors.Breast Cancer Res Treat. 1995; 36: 237-251Crossref PubMed Scopus (119) Google Scholar, 8Griffioen AW Coenen MJ Damen CA Hellwig SM van Weering DH Vooys W Blijham GH Groenewegen G CD44 is involved in tumor angiogenesis: an activation antigen on human endothelial cells.Blood. 1997; 90: 1150-1159Crossref PubMed Google Scholar, 9van Beijnum JR Dings RP van der Linden E Zwaans BM Ramaekers FC Mayo KH Griffioen AW Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature.Blood. 2006; 108: 2339-2348Crossref PubMed Scopus (224) Google Scholar Recently, we identified galectin-1 as a target molecule in the tumor endothelium. Activated tumor endothelial cells increase the expression of galectin-1, and ablation of its expression in vitro and in vivo results in impaired endothelial cell function and hampered tumor angiogenesis.10Thijssen VL Postel R Brandwijk RJ Dings RP Nesmelova I Satijn S Verhofstad N Nakabeppu Y Baum LG Bakkers J Mayo KH Poirier F Griffioen AW Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy.Proc Natl Acad Sci USA. 2006; 103: 15975-15980Crossref PubMed Scopus (404) Google Scholar Whether other galectins are also directly involved in endothelial cell biology and tumor angiogenesis is less well studied (for review, see 11Thijssen VL Poirier F Baum LG Griffioen AW Galectins in the tumor endothelium: opportunities for combined cancer therapy.Blood. 2007; 110: 2819-2827Crossref PubMed Scopus (115) Google Scholar). In the current study, we analyzed the expression of all known human galectins in the endothelium to get more insight in their possible function in endothelial cell biology. We show that multiple galectins and galectin isoforms are expressed by endothelial cells. Furthermore, the expression and distribution of endothelial galectins is altered on cell activation. These observations provide prospects for the development of therapies that target galectins in the tumor endothelium. Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords by infusion of trypsin into the vein and incubation for 20 minutes at 37°C. Cells were collected in HUVEC culture medium [RPMI (Invitrogen, Breda, the Netherlands) containing 10% fetal calf serum (Invitrogen) and 10% human serum and supplemented with l-glutamin (Invitrogen) and penicillin/streptomycin (Invitrogen)] by perfusion with PBS and divided over two T25 cultured flasks coated with 0.2% gelatin/PBS. After incubation for 1 hour at 37°C/5% CO2, unattached cells and erythrocytes were removed by washing three times with PBS. Next, the cells in one flask were directly collected and lysed for RNA isolation, Western blot, or fluorescence-activated cell sorting (FACS) analysis, and the other cells were passaged every 3 to 5 days and kept in HUVEC culture medium at 37°C and 5% CO2 up to three passages. For additional in vitro cell activation, HUVECs were cultured for 3 days in HUVEC culture medium supplemented with 10% culture medium of both colon carcinoma cell lines CaCo2 and LS174. The origin and culture conditions of all endothelial cell lines have been described previously.12van Beijnum JR van der Linden E Griffioen AW Angiogenic profiling and comparison of immortalized endothelial cells for functional genomics.Exp Cell Res. 2008; 314: 264-272Crossref PubMed Scopus (42) Google Scholar All tissues, frozen or paraffin embedded, were obtained from the Maastricht Pathology Tissue Collection (Maastricht, the Netherlands). Collection, storage, and use of tissue and patient data were performed in agreement with the Code for Proper Secondary Use of Human Tissue in the Netherlands. Total RNA was isolated using the RNeasy kit (QIAgen, Venlo, the Netherlands) according to the suppliers protocol from freshly isolated or cultured cells or frozen tissue sections (10- × 20-μm sections). All isolations were subjected to on-column DNase treatment (QIAgen) to remove any genomic DNA contaminations. The concentration and purity of the RNA was analyzed using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed with the iScript cDNA synthesis kit (Bio-Rad, Veenendaal, the Netherlands) on 100 ng of RNA according to the suppliers protocol. After cDNA synthesis, nuclease-free water was added up to a final volume of 50 μl. Real-time PCR was performed on the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) using a standard two-step amplification protocol (Ta at 60°C) followed by a melting curve analysis. For each reaction, 1.5 μl of cDNA was used in a total volume of 25 μl containing 1× iQ SYBR Green supermix (Bio-Rad) and 400 nmol/L of the appropriate forward and reverse primer. Each PCR was performed in duplicate on separate plates. Primers were designed to target specifically to human galectins as described previously.13Thijssen VL Brandwijk RJ Dings RP Griffioen AW Angiogenesis gene expression profiling in xenograft models to study cellular interactions.Exp Cell Res. 2004; 299: 286-293Crossref PubMed Scopus (73) Google Scholar All primers were synthesized by Eurogentec (Liège, Belgium). For Western blotting, cultured cells were directly lysed in the culture flask using 2× sample buffer (100 mmol/L Tris, pH 6.8, 200 mmol/L dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol). Samples were boiled for 5 minutes and quenched on ice. Subsequently, proteins were separated on a 15% polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes (Schleicher & Schuell, Den Bosch, the Netherlands) according to standard procedures. Equal protein loading was confirmed by Ponceau red staining. Membranes were blocked with 5% nonfat dry milk (Bio-Rad) in 0.1% Tween 20/PBS and incubated overnight at 4°C with rabbit anti-galectin-1 antibody (dilution 1:500; kind gift of Dr. L.G. Baum, University of California, Los Angeles), goat anti-galectin-3 antibody (0.2 ng/ml; R&D Systems, Abingdon, UK), goat anti-galectin-8 antibody (0.2 ng/ml; R&D Systems), or goat anti-galectin-9 antibody (0.2 ng/ml; R&D Systems). After three washes with PBS for 5 minutes, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Finally, membranes were washed three times for 5 minutes with PBS, and staining was visualized using 3′,3′-diaminobenzidine (0.5 mg/ml in 0.05 mol/L Tris-HCl, pH 7.6, supplemented with 0.03% H2O2). For FACS analysis, freshly isolated, cultured, and tumor-conditioned HUVECs were harvested and fixed with 1% paraformaldehyde for 20 minutes at room temperature. Cells were washed in 0.1% BSA, 0.01% sodium azide, and PBS, incubated on ice with the appropriate anti-galectin antibody [rabbit anti-galectin-1, 1:1000 (gift from Dr. L.G. Baum); goat anti-galectin-3, 1:30 (R&D Systems); goat anti-galectin-8, 1:30 (R&D Systems); goat anti-galectin-9, 1:30 (R&D Systems)], and diluted in 0.1% BSA, 0.01% sodium azide, and PBS in the presence or absence of 0.05% Triton-100. Subsequently, cells were washed with PBS and incubated with an appropriate PE-labeled secondary antibody, washed with PBS, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Breda, the Netherlands). All experiments were performed on five different HUVEC isolations. For cytospins, cultured HUVECs were harvested and stained similar to FACS analysis. After the staining procedure, the cells were centrifuged for 5 minutes at 700 rpm onto standard glass microscope slides. Finally, the cells were mounted in Mowiol (Hoechst, Frankfurt, Germany) supplemented with 4′,6-diamidino-2-phenylindole (Invitrogen) for nuclear staining. Immunohistochemistry was performed on paraffin-embedded tissue sections. Staining for CD31/34 and galectin-1 were performed as described previously.10Thijssen VL Postel R Brandwijk RJ Dings RP Nesmelova I Satijn S Verhofstad N Nakabeppu Y Baum LG Bakkers J Mayo KH Poirier F Griffioen AW Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy.Proc Natl Acad Sci USA. 2006; 103: 15975-15980Crossref PubMed Scopus (404) Google Scholar For galectin-3, -8, and -9 staining, sections were dewaxed and incubated in 0.3% H2O2/methanol. Next, the sections were microwave pretreated in citric acid and blocked with 1% BSA/PBS. Primary antibodies [goat anti-galectin-3, 1:30 (R&D Systems); goat anti-galectin-8, 1:30 (R&D Systems); and goat anti-galectin-9, 1:30 (R&D Systems)] were applied in 0.5% BSA/PBS. Finally, biotin-labeled secondary antibody was applied, and staining was performed using the StreptABComplex/horseradish peroxidase kit (Dako, Heverlee, Belgium). The sections were counterstained with hematoxylin (Merck, Haarlem, the Netherlands), dehydrated, and mounted in Entellan (Merck). Data of quantitative PCR (qPCR) are shown as mean values ± SD. FACS data are presented as mean values ± SEM. The Mann-Whitney rank sum test was used to calculate statistically significant differences in mRNA expression, protein expression, or protein localization on cell activation. P values <0.05 were considered statistically significant, and all calculations were performed in SPSS 12.0.1. (SPSS Inc., Gorinchem, the Netherlands). In this study, we examined galectin expression in endothelial cells. Up to now, 15 galectins have been described in literature, 11 of which are also expressed in humans (Table 1). Because little is known about their expression in endothelial cells, qPCR primers and full-length cDNA primers were designed for all 11 human galectin family members (Table 2). To validate the qPCR analysis, the primers were tested on available cDNA clones or on cDNA derived from tissues/cell lines that have been reported to express a specific galectin. The different amplicons were analyzed by gel electrophoresis and cloned into pCR2.1. To confirm primer specificity, the clones were sequenced, and PCR was performed with the appropriate primers (Figure 1A). Next, the cloned PCR fragments were used to determine primer sensitivity. For this, dilution series were generated with the cloned PCR fragments that covered a concentration range of at least six logscales (Figure 1B). All qPCR primers displayed an optimal amplification slope of approximately −3.3 and a broad cycle threshold range in which there was linear amplification (Table 3).Table 1List of All Known Human GalectinsNameENSEMBLE accession numberType*Prototype galectins consist of a single carbohydrate recognition domain. Tandem repeat galectins are composed of two carbohydrate recognition domains connected by a linker peptide. Chimera galectins contain a proline/glycine rich tail at the N-terminus of a single carbohydrate recognition domain.LGALS1ENSG00000100097PrototypeLGALS2ENSG00000100079PrototypeLGALS3ENSG00000131981ChimeraLGALS4ENSG00000171747Tandem repeatLGALS7ENSG00000178934PrototypeLGALS8†Splicing has been shown to occur in sequence encoding the linker peptide between the two carbohydrate recognition domains.ENSG00000116977Tandem repeatLGALS9†Splicing has been shown to occur in sequence encoding the linker peptide between the two carbohydrate recognition domains.ENSG00000168961Tandem repeatLGALS10ENSG00000105205PrototypeLGALS12ENSG00000133317Tandem repeatLGALS13ENSG00000105198PrototypeLGALS14†Splicing has been shown to occur in sequence encoding the linker peptide between the two carbohydrate recognition domains.ENSG00000006659Prototype* Prototype galectins consist of a single carbohydrate recognition domain. Tandem repeat galectins are composed of two carbohydrate recognition domains connected by a linker peptide. Chimera galectins contain a proline/glycine rich tail at the N-terminus of a single carbohydrate recognition domain.† Splicing has been shown to occur in sequence encoding the linker peptide between the two carbohydrate recognition domains. Open table in a new tab Table 2Galectin Primers Used for Full-Length PCR or qPCRFull-length primersqPCR primersgal-1 F*F, forward primer; R, reverse primer.5′-ATGGCTTGTGGTCTGGTC-3′gal-1 F5′-TGCAACAGCAAGGACGGC-3′gal-1 R5′-TCAGTCAAAGGCCACACA-3′gal-1 R5′-CACCTCTGCAACACTTCCA-3′gal-2 F5′-ATGACGGGGGAACTTGAG-3′gal-2 F5′-GATGGCACTGATGGCTTTG-3′gal-2 R5′-TTATTCTTTTAACTTGAAAGAGGA-3′gal-2 R5′-AGACAATGGTGGATTCGCT-3′gal-3 F5′-ATGGCAGACAATTTTTCG-3′gal-3 F5′-CAGAATTGCTTTAGATTTCCAA-3′gal-3 R5′-TTATATCATGGTATATGAAGCAC-3′gal-3 R5′-TTATCCAGCTTTGTATTGCAAgal-4 F5′-ATGGCCTATGTCCCCGCA-3′gal-4 F5′-CGAGGAGAAGAAGATCACCC-3′gal-4 R5′-TTAGATCTGGACATAGGACAAGG-3′gal-4 R5′-CTCTGGAAGGCCGAGAGG-3′gal-7 F5′-ATGTCCAACGTCCCCCAC-3′gal-7 F5′-CAGCAAGGAGCAAGGCTC-3′gal-7 R5′-TCAGAAGATCCTCACGGA-3′gal-7 R5′-AAGTGGTGGTACTGGGCG-3′gal-8 F5′-AGAATGATGTTGTCCTTAAAC-3′gal-8 F5′-CTTAGGCTGCCATTCGCT-3′gal-8 R5′-CTACCAGCTCCTTACTTCC-3′gal-8 R5′-AAGCTTTTGGCATTTGCA-3′gal-9 F5′-ATGGCCTTCAGCGGTTCC-3′gal-9 F5′-CTTTCATCACCACCATTCTG-3′gal-9 R5′-CTATGTCTGCACATGGGTCAG-3′gal-9 R5′-ATGTGGAACCTCTGAGCACTG-3′gal-10 F5′-ATGTCCCTGCTACCCGTG-3′gal-10 F5′-AGTGTGCTTTGGTCGTCGT-3′gal-10 R5′-TTATCTCTTTAAATAGCTGACAT-3′gal-10 R5′-ATGCTCAGTTCAAATTCTTGG-3′gal-12 F5′-ATGAGTCAGCCCAGTGGG-3′gal-12 F5′-TGTGAGCCTGAGGGACCA-3′gal-12 R5′-TCAGGAGTGGACACAGTAGAG-3′gal-12 R5′-GCTGAGATCAGTTTCTTCTGC-3′gal-13 F5′-ATGTCTTCTTTACCCGTG-3′gal-13 F5′-CTTTACCCGTGCCATACAA-3′gal-13 R5′-TCAATTGCAGACACACACT-3′gal-13 R5′-GTGGGTCATTGATAAAAGAGTG-3′gal-14 F5′-ATGTCCCTGACCCACAG-3′gal-14 F5′-CCTTGATGATTGTGGTACCAT-3′gal-14 R5′-TCAATCGCTGATAAGCACT-3′gal-14 R5′-GTGGGTCCTTGACAAAAGTG-3′* F, forward primer; R, reverse primer. Open table in a new tab Table 3Primer SensitivityTarget geneSlope (SEM)Lower limit (SEM)Upper limit (SEM)LGALS13.36 (0.08)14.67 (3.18)31.00 (1.15)LGALS23.27 (0.03)16.67 (2.33)31.67 (1.67)LGALS33.10 (0.16)10.33 (0.33)30.00 (0.58)LGALS43.35 (0.11)12.67 (0.33)32.00 (1.53)LGALS73.28 (0.15)13.00 (0.58)33.00 (2.31)LGALS83.25 (0.07)14.33 (2.85)25.67 (1.86)LGALS93.27 (0.15)13.33 (2.40)33.33 (1.33)LGALS103.20 (0.12)10.33 (0.33)29.00 (2.31)LGALS123.47 (0.10)13.00 (1.00)33.67 (0.88)LGALS133.10 (0.15)13.67 (1.20)33.33 (0.88)LGALS143.04 (0.04)13.33 (1.76)29.67 (0.33) Open table in a new tab To determine the galectin mRNA expression levels in endothelial cells, we next used the primers to perform qPCR on cDNA generated from quiescent HUVECs immediately after isolation from the umbilical vein. Results show that these endothelial cells mainly express galectin-1, -3, -8, and -9 (Figure 1C). In addition, faint expression of galectin-2, -4, and -12 could be detected (Figure 1C, inset). Galectin-7, -10, -13, and -14 all had cycle threshold values that were above the upper limit of linear amplification, and these galectins were therefore considered to be not expressed in quiescent endothelial cells. To compare the galectin expression profile in endothelial cells of different origin, we also performed qPCR on cDNA derived from a human microvascular endothelial cell line. There was a strong correlation (r = 0.967, P < 0.001) between galectin expression in cultured HUVECs and human microvascular endothelial cells (Figure 1D). Similar correlations were observed when expression in HUVECs was compared with expression in endothelial cell lines RF24 (r = 0.818, P = 0.004) and EVLc2 (r = 0.879, P = 0.001). These data indicate that cultured endothelial cells of different origin express a similar repertoire of galectins. For further analysis, we focused galectin-1, -3, -8, and -9, given the low expression of the other galectins in all endothelial cells tested. We used primers spanning the entire coding sequence to identify possible splice variants. For galectin-1 and -3, a single transcript was detected of the expected size, 450 and 750 bp, respectively. In contrast, multiple bands of different lengths were observed for galectin-8 (950 and 1150 bp) and galectin-9 (1050, 1200, and 1350 bp) (Figure 1E). Current research is focusing on the functional relevance of the different splice variants. To confirm expression at the protein level, several approaches were followed. First, Western blotting was performed on total cell lysates. In line with the PCR data, protein expression could be detected for galectin-1, -3, -8, and -9 (Figure 1F). Galectin-1 showed an intense band at the expected molecular weight of 14 kDa. Galectin-3 displayed one prominent band at 25 kDa and another faint band at 35 kDa. Galectin-8 protein was hardly detectable, but three faint bands were visible at 34, 36, and 38 kDa. For galectin-9, one band was visible at 37 kDa together with a small band of 14 kDa. Cross-reactivity of the galectin-9 antibody with galectin-1 was excluded in a Western blot using recombinant human galectin-1 protein, which only displayed a 14-kDa band when the galectin-1 antibody was used (data not shown). Second, protein expression was detected using FACS. Again, galectin-1, -3, -8, and -9 were readily detectable (data shown below). Third, to get more insight in galectin protein localization, immunohistochemical staining was performed on cultured endothelial cells. Whereas the endothelial cell-specific marker CD31, which was used as positive control, showed strong staining, all four galectins showed a clear, dot-like staining pattern, further confirming their endothelial expression (Figure 1G). All of these results show that quiescent endothelial cells in vitro express a broad panel of galectins with galectin-1, -3, -8, and -9 as the most prominent ones. To study the endothelial expression in vivo, immunohistochemical staining was performed on different human tissues. Vessels in these tissues were identified using an endothelial cell staining with anti-CD31/34 antibodies (Figure 2, A–D). Galectin-1 staining was observed in the endothelial cells of all tissues (Figure 2, E–H). In all tissues, faint staining could be detected throughout the cell (Figure 2, E–H). The staining of galectin-3 was less constant (Figure 2, I–L). Although no galectin-3 was detectable in the endothelial cells of liver (Figure 2J), faint staining could occasionally be observed in vessels of kidney and placenta both in the cytoplasm and in the nucleus (Figure 2, I and K). In colon, all endothelial cells displayed weak galectin-3 staining throughout the cell (Figure 2L). Endothelial galectin-8 staining was only rarely observed. Apart from colon (Figure 2P) and a sporadic faint staining in kidney (Figure 2O), galectin-8 was undetectable in the vessels of most tissues (Figure 2, M and N). Vascular staining was also undetectable for galectin-9 in kidney (Figure 2S). In placenta endothelium, sporadic nuclear staining could be observed (Figure 2Q). The same was true for liver (Figure 2R), whereas nuclear galectin-9 staining in endothelial cells was common in colon (Figure 2T). All of these data suggest that besides a common expression of galectin-1 in the endothelial cells of normal tissues, the expression of endothelial galectin-3, -8, and -9 protein is more variable and depends on the environment/tissue surrounding the endothelial cell. Because culturing endothelial cells under high serum conditions has been reported to increase galectin-1 expression,10Thijssen VL Postel R Brandwijk RJ Dings RP Nesmelova I Satijn S Verhofstad N Nakabeppu Y Baum LG Bakkers J Mayo KH Poirier F Griffioen AW Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy.Proc Natl Acad Sci USA. 2006; 103: 15975-15980Crossref PubMed Scopus (404) Google Scholar we next performed qPCR to compare the galectin mRNA expression between freshly isolated endothelial cells and cells cultured in the presence of 20% human serum. In agreement with our previous observations, cells cultured in 20% human serum significantly induce galectin-1 mRNA expression compared with quiescent cells (5.32 ± 1.97-fold; P = 0.04) (Figure 3A). In fact, galectin-1 was the only galectin of which the expression increased. Of the other abundantly expressed galectins, galectin-3 mRNA expression did not significantly change (1.91 ± 0.80; P = ns) whereas galectin-8 and -9 expression significantly decreased, respectively, 0.59 ± 0.12-fold (P < 0.04) and 0.32 ± 0.06-fold (P < 0.002). The expression of galectin-4, which was already low, further decreased (0.36 ± 0.20-fold, P = 0.04), and all of the other galectins were undetectable (data not shown). Additional activation by culturing the endothelial cells in the presence of basic fibroblast growth factor (data not shown) or tumor-conditioned medium (Figure 3B) did not further increase the expression of galectin-1 or affect the expression of galectin-3. The expression of galectin-8 and -9 was also not further decreased, suggesting that the changes in expression are an early event in endothelial cell activation. To test whether the alterations in mRNA expression were also reflected at the protein level, quiescent, cultured, and tumor-activated endothelial cells were subjected to flow cytometry. To distinguish between total and membrane-bound galectin proteins, the paraformaldehyde-fixed cells were incubated with the primary antibody in the presence (total protein) or absence (membrane-bound protein) of the permeabilization agent Triton. Using this approach, it could be observed that approximately 30% of galectin-1 and -9 protein was located extracellularly (Figure 3C). For galectin-8, almost one-half of the total protein content was located at the outer surface of the cell, whereas galectin-3 appeared to be exclusively present at the cell membrane. When the effects of cell activation on total galectin protein content were measured, a similar trend was observed as for mRNA expression, albeit not significant (Figure 3D). Only galectin-8 protein levels appeared to be unaltered in contrast to the significant decrease in mRNA expression. Interestingly, the amount of membrane-bound protein showed a more prominent increase, except for galectin-3 (Figure 3E). After culturing the cells in 20% serum, the fluorescence intensity of extracellular galectin-1 significantly increased almost sixfold (P = 0.005), galectin-8 increased threefold (P = 0.002), and even galectin-9 signals significantly increased (1.4-fold, P = 0.04) despite the trend toward decreased total protein levels. Again, additional activation with basic fibroblast growth factor or tumor-conditioned medium did not affect the expression, although the increase in galectin-8 was partially reversed. These observations suggested that on endothelial cell activation, galectin-1, -8, and -9 were translocated to the extracellular compartment. Calculation of the ratio of between extracellular and total galectin protein levels showed significantly increased membrane localization for galectin-1 and -9 (Figure 3F). The results above indicate that the endothelial expression and localization of galectins in vitro is altered on cell activation. We have previously shown that galectin-1 expression is increased in the activated endothelial cells of different human tumors, including colon and breast carcinoma as well as Ewing sarcoma.10Thijssen VL Postel R Brandwijk RJ Dings RP Nesmelova I Satijn S Verhofstad N Nakabeppu Y Baum LG Bakkers J Mayo KH Poirier F Griffioen AW Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy.Proc Natl Acad Sci USA. 2006; 103: 15975-15980Crossref PubMed Scopus (404) Google Scholar Thus, we next studied the expression of galectins in the endothelium of colon tumor tissue using immunohistochemical staining. Similar as described above, CD31/34 staining was used to identify the endothelial cells (Figure 4A). Again, a clear increase in galectin-1 staining intensity could be observed in the tumor endothelial cells (Figure 4B) compared with normal tissue (Figure 2H). Galectin-3 staining remained lightly positive and could be observed throughout the cell (Figure 4C). For galectin-8 and -9, more subtle changes could be observed. In normal tissue, the expression of both galectins was mainly detectable in the nuclei of endothelial cells. In tumor tissue, the number of positive cells appeared to decrease. Whereas the staining of galectin-9 in the positive cells was also detected more regularly in the cytoplasm, galectin-8 expression was only detectable in the nucle