Brain Advance Access originally published online on August 9, 2008
Brain 2008 131(10):2579-2595; doi:10.1093/brain/awn182
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VEGF-dependent induction of CD62E on endothelial cells mediates glioma tropism of adult haematopoietic progenitor cells
1Department of General Neurology, Laboratory of Molecular Neurooncology, Hertie Institute for Clinical Brain Research, 2Institute of Brain Research, University of T übingen, T übingen, Germany, 3Institute of Neuropathology, University of Zurich, Switzerland, 4Department of Neurosurgery, Klinikum Großhadern, Ludwig-Maximilians University of Munich, Munich, 5Department of Internal Medicine II (Hematology), University of T übingen, T übingen, Germany, 6Department of Neurology, University of Zurich, Switzerland and 7Clinical Cooperation Unit Neurooncology, German Cancer Research Center, Heidelberg, Germany
Correspondence to: Ghazaleh Tabatabai, MD, Department of General Neurology, Laboratory of Molecular Neurooncology, Hertie Institute for Clinical Brain Research, University of T übingen, Hoppe-Seyler-Strasse 3, 72076 T übingen, Germany E-mail: ghazaleh.tabatabai{at}uni-tuebingen.de
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Summary |
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Haematopoietic progenitor cells (HPC) are attracted by experimental gliomas in vivo. This attraction is further enhanced by irradiation or hypoxic preconditioning of the glioma cells. Adhesive interactions might be critical to the preferential accumulation of HPC within the glioma tissue. Here, we studied the interactions of HPC with endothelial cells. Exposure of human cerebral endothelial cells (SV-HCEC), human microvascular endothelial cells (HMEC) and brain tumour endothelial cells derived from human glioblastomas (BTEC) to supernatants of glioma cells and primary glioma cells (SN-G) induced the expression of E-selectin (CD62E). CD62E expression was further enhanced when the glioma cells had been exposed to irradiation or hypoxia prior to the collection of supernatants, as well as by irradiation or exposure to hypoxia of the endothelial cells. Vascular cell adhesion molecule 1 (VCAM-1) was constitutively expressed on SV-HCEC, HMEC and BTEC, but was not modulated by SN-G, irradiation or hypoxia. Transendothelial HPC migration was enhanced after CD62E induction in vitro. Neutralizing antibodies to CD62E strongly reduced the homing of lin–Sca-1+c-kit+ cells to orthotopic SMA-560 gliomas in vivo. Tissue microarray sampling normal brain tissue and astrocytomas of WHO grades II–IV revealed a selective expression of CD62E on endothelial cells of tumour vessels. SN-G-induced CD62E expression on endothelial cells in vitro required transforming growth factor (TGF)-β signalling in glioma cells and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGF-R2) signalling in endothelial cells. Further, we observed a nuclear factor kappa B-dependent activation of the CD62E promoter peaking at 12 h after VEGF-R2 activation by glioma-derived VEGF. Taken together, we identify glioma cell-induced CD62E expression on endothelial cells as one mediator of the glioma tropism of HPC.
Key Words: brain tumour; haematopoietic progenitor cells; hypoxia; irradiation; vascular endothelial growth factor, CD62E
Abbreviations:
BTEC, brain tumour endothelial cells isolated from human glioblastoma tissue; CD, cluster of differentiation; CD62E, E-selectin; CD62P, platelet selectin; CXCL12, CXC chemokine ligand 12; DAPI, 4',6-diamidino-2-phenylindole; ELISA, Enzyme-linked immunosorbent assay; FCS, foetal calf serum; G-CSF, granulocyte colony stimulating factor; HIF, hypoxia-inducible factor; HMEC, human microvascular endothelial cells; HPC, haematopoietic progenitor and stem cells; IL, interleukin; LSK, lin–Sca-1+c-kit+; NF
B, nuclear factor kappa B; NT, no treatment; pVEGF-R2, phosphorylated VEGF-R2; sKitL, soluble Kit ligand; sCD62E, soluble E-selectin; SCF, stem cell factor; SDF, stromal cell-derived factor; SN-G, supernatant of glioma cell lines LNT-229, LN-308 and primary glioma cultures T113, T132, T159; SN-Gp, supernatant of LNT-229 puro cells; SN-GpSD-208, supernatant of SD-208-treated LNT-229 puro cells; SN-G-siTGF-β, supernatant of LNT-229 siTGF-β1,2 cells stably expressing shRNAs targeting TGF-β1 and TGF-β2; SN-FHAS, supernatant of SV-FHAS cells; SN-GRT, supernatant of irradiated glioma cells; SN-GHO, supernatant of hypoxic glioma cells; SFM, serum-free medium; SFI, specific fluorescence index; SV-HCEC, human cerebral endothelial cells; SV-FHAS, human astrocytic cell line; TGF-β, transforming growth factor-β; TNF-
, tumour necrosis factor-; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VEGF-R, VEGF receptor
Received December 18, 2007. Revised July 11, 2008. Accepted July 17, 2008.
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Introduction |
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Intracerebral experimental gliomas attract intravenously injected CD34+ haematopoietic progenitor and stem cells (HPC). The underlying molecular pathways leading to the glioma tropism of HPC involve a transforming growth factor (TGF)-β-dependent signalling cascade regulating the release of stromal cell-derived factor (SDF)-1
/CXC chemokine ligand 12 (CXCL12) by glioma cells. CXCL12 interacts with the chemokine receptor CXCR4 on HPC. Further, matrix metalloproteinase 9 cleaves and thereby releases soluble Kit ligand (sKitL), which interacts with the receptor CD117 on HPC (Tabatabai et al., 2005
). Cerebral irradiation promotes the homing of HPC to the tumour bulk and to tumour satellites in vivo. Supernatants of irradiated or hypoxic glioma cells enhance HPC migration in vitro due to a TGF-β-dependent, hypoxia-inducible factor-1 (HIF-1)-mediated induction of CXCL12 promoter activity in glioma cells (Tabatabai et al., 2006). CXCL12 is also an important mediator for the attraction of mesenchymal stem cells to experimental gliomas (Nakamizo et al., 2005). A systemic gradient of CXCL12 alone, however, does not explain the glioma-mediated attraction of intravenously injected HPC. Moreover, further insight into the mechanism of the glioma tropism is required for the design of a HPC-based cellular therapy that specifically targets gliomas.
The regional expression of endothelial adhesion molecules may contribute to the specificity of HPC homing towards gliomas. Similarities in the trafficking and homing of HPC and leucocytes to particular tissue sites arise. The diapedesis of leucocytes to sites of infection and inflammation is an important step in their recruitment. This process ensures appropriate innate and adaptive immunological responses. Adhesion molecules mediate rolling and arrest of leucocytes on the vessel wall as key steps prior to the transmigration across the endothelial layer (Millan et al., 2006; Nieminen et al., 2006).
Adhesion is also critical for the interaction of HPC within the haematopoietic microenvironment in the bone marrow. Important mediators of this adhesive interaction include vascular cell adhesion molecule 1 (VCAM-1), platelet selectin (CD62P) and endothelial selectin (CD62E) (Frenette et al., 1998; Katayama et al., 2003). CD62P is deposited in storage granules of platelets and endothelial cells. After activation, it is translocated to the surface (Bevilacqua et al., 1989). VCAM-1 and CD62E are solely expressed on endothelial cells (Mazo et al., 1998). In this study, we analysed the role of HPC adhesion to endothelial cells in the glioma tropism of HPC.
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Material and Methods |
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Cell culture
Human adult HPC were isolated by anti-CD34 immunomagnetic microbeads (Bautz et al., 2001
). After informed consent, peripheral blood cells were obtained from healthy donors or patients with non-haematological malignancies during G-CSF-induced stem cell mobilization, according to the guidelines of the ethics committee of the University of Tübingen (project number 268/2003-LP). The human CD34 antigen is a reliable marker for identifying a small fraction of human bone marrow and peripheral blood mononuclear cells consisting of primitive uncommitted and pluripotent haematopoietic progenitor cells as well as haematopoietic stem cells (Andrews et al., 1986; Berenson et al., 1988; Terstappen et al., 1991; Van Epps et al., 1994; Gothot et al., 1998; Gao et al., 2001; Albo et al., 2004). The source, generation and culture conditions of the cerebral vascular endothelial cell line SV-HCEC and the astrocytic cell line SV-FHAS have been described (Muruganandam et al., 1997). Brain tumour endothelial cells (BTEC) were isolated from freshly resected human glioblastomas and characterized for their endothelial cell phenotype as described (Miebach et al., 2006). Briefly, the tissue was minced, homogenized and digested enzymatically. The cells were separated by percoll gradient centrifugation. CD31-positive cells were isolated by magnetic beads (Dynal, Hamburg, Germany). Only passage 0–2 cells were used for the experiments. Human microvascular endothelial cells (HMEC) were purchased from Cascade Biologics (Invitrogen GmbH, Karlsruhe, Germany). All glioma cell lines were kindly provided by Dr N. de Tribolet (Lausanne, Switzerland). In contrast to other LN-229 sublines, LN-229 glioma cells cultured in our laboratory exhibit wild-type p53 status (Wischhusen et al., 2003) and are designated LNT-229. The generation of LNT-229 sublines stably depleted from TGF-β1 to TGF-β2 by RNA interference (LNT-229 siTGF-β1,2) and the control cells (LNT-229 puro) has been described (Friese et al., 2004). The following primary glioma cultures were used: T113, T159, derived from glioblastomas and T132, derived from an anaplastic astrocytoma. The primary human glioma cultures were established from samples with high tumour content (Bähr et al., 2003). Supernatants of glioma cells (SN-G) were generated in serum-free medium (SFM): 1.5 million glioma cells were first seeded in serum-containing medium in T75 cell culture flasks. The medium was removed on the following day and the cells were washed three times with PBS. Seven millilitres SFM were added, 48 h later, supernatants were harvested. Supernatant was concentrated using a Centriplus centrifugal filter device YM-3 (3 kD cut-off, Millipore, Eschborn, Germany). Protein quantification was performed using the Bradford assay (BioRad, Munich, Germany). Treatment of endothelial cells with SN-G was performed for 6, 12, 24, 48 or 96 h. Incubation of cells in hypoxia was performed at 37°C and 1% O2 for 6, 12, 24, 48 or 96 h (Wick et al., 2002a). Cells were irradiated at 2 or 8 Gy 
-cell, Nordion, Kanata, CA, USA) and analysed at 6, 12, 24, 48 or 96 h thereafter (Wick et al., 2002b). Recombinant TGF-β2 and vascular endothelial growth factor (VEGF165), neutralizing antibodies to VEGF (clone 26503) and VEGF-R2 (clone 89106) were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). SD-208 was provided by Scios (Fremont, CA, USA) (Uhl et al., 2004). siRNA pools targeting human TGF-β1, TGF-β2 or VEGF containing four selected siRNA duplexes each with a modification pattern that addresses off-target effects caused by both strands (ON-TARGETplus SMARTpool) and the non-targeting control pool (ON-TARGETplus siCONTROL Non-targeting Pool) were designed by and purchased from Dharmacon (Lafayette, CO, USA). The pool of siRNA duplexes targeting TGF-β1 had the following sense sequences: 5'-AUUGAGGGCUUUCGCCUUAUU-3', 5'-CCGAGAAGCGGUACCUGAAUU-3', 5'-GCAGAGUACACACAGCAUAUU-3', 5'-GTGACUAUCCACCUGCAAGAUU-3'. The siRNA duplexes targeting TGF-β2 had the following sense sequences: 5'-GGAUUGAGCUAUAUCAGAUUU-3', 5'-CUGCGUGUCCCAAGAUUUAUU-3'; 5'-GAUGCGGCCUAUUGCUUUAUU-3', 5'-GAGCAUGCCCGUAUUUAUGUU-3'. The siRNA duplexes targeting VEGF had the following sense sequences: 5'-GCAGAAUCAUCACGAAGUG, 5'-CAACAAAUGUGAAUGCAGA, 5'-GGAGUACCCUGAUGAGAUC, 5'-GAUCAAACCUCACCAAGGC. Lipophilic transfection was performed with Metafectene Pro (Biontex, Martinsried, Germany).
Transendothelial migration of HPC
Migration assays were performed in transwell plates (Costar, Cambridge, MA, USA) of 6.5 mm diameter with 5 µm pore filters. SV-HCEC, HMEC or BTEC were plated at 105 cells/transwell on filters coated with fibronectin. Non-adherent cells were removed after 24 h. The adherent cells were cultured for 48 h to obtain confluent endothelial monolayers. Confluency of the endothelial monolayers was confirmed by measuring permeability for albumin (Möhle et al., 1997,1998; Naiyer et al., 1999). The monolayers were used unstimulated, stimulated with SN-G or after exposure to irradiation or hypoxia. Prior to the migration experiment, the monolayer was washed three times with PBS. The 105 CD34+ HPC in 200 µl SFM were added to the upper compartment. SFM, recombinant CXCL12 or SN-G were added to the lower compartment. In neutralization experiments, the SV-HCEC were pre-incubated for 4 h at 37°C with -CD62E (R&D, clone BBIG-E4[5D11]) or -VCAM-1 (R&D, clone BBIG-V1 [4B2]) or appropriate control antibodies. Migration was assessed at 37°C, 5% CO2 for 16 h. Cells that had migrated to the lower compartment were quantified with the CyQuant Assay (Invitrogen, Karlsruhe, Germany).
Reporter assays
For dual luciferase/renilla assays (Dyer et al., 2000) 200 ng of the respective reporter constructs was cotransfected with 20 ng of pRL-CMV. pGL3b.CD62E contains the full-length promoter of CD62E (Nübel et al., 2004). pNFB-Luc contains a firefly luciferase gene that is especially designed for the detection of nuclear factor kappa B (NFB) signal transduction pathway activity. It contains four copies of the NFB consensus sequence fused to a TATA-like promoter region from the Herpes simplex virus thymidine kinase promoter. The binding of endogenous NFB leads to induction of transcription and activation of the reporter gene in the transfected cells indicating transcriptional activity of NFB. Ad-IkBM, containing the NFB repressor, and the control Ad-Mock were kindly provided by Dr P. A. Baker (McGill University, Ottawa, Canada).
TransAM NFB family
Nuclear extracts were prepared (Nuclear Extract Kit, Active Motif, Rixensat, Belgium) from SV-HCEC, HMEC or BTEC. Raji extracts, provided by the assay, served as positive control. Nuclear extracts from NIH3T3 fibroblasts served as negative control. The transcriptional activity of the NFB family p50, p65, p52, c-Rel and RelB was analysed by the transcripton factor assay TransAM NFB family (Active Motif) according to manufacturer's protocol. Briefly, this assay is based on an enzyme-linked immunosorbent assay (ELISA) format using 96-well plates coated with immobilized oligonucleotides containing the NFB consensus site. The active form of NFB contained in nuclear extracts of cells binds to the oligonucleotides. Primary antibodies recognizing epitopes on activated p50, p52, p65, c-Rel or RelB and HRP-conjugated secondary antibodies are used for the detection and quantification of active NFB family members by spectrophotometry.
Immunoblot
Immunoblots were performed as described (Tabatabai et al., 2006). The following antibodies were used: phospho-Smad 2 (Ser 465/467), total Smad 2 (Cell Signaling, Boston, MA, USA), NFB p50 and p65 (Santa Cruz, CA, USA) and GAPDH (Chemicon, Billerica, MA, USA).
Enzyme-linked immunosorbent assay
The concentrations of VEGF, sCD62E, TGF-β1 and TGF-β2 in the supernatants were determined by Quantikine Immunoassays from R&D Systems. A sandwich ELISA was performed to measure the levels of phosphorylated VEGF-R2 and total VEGF-R2 in cell lysates (Duo Set IC, R&D Systems). The proliferation of cells was assessed by a cell proliferation biotrak ELISA system (GE Healthcare, Buckinghamshire, UK).
Flow cytometry
SV-HCEC, HMEC or BTEC were grown in 12-well plates either untreated or stimulated with the indicated supernatant, hypoxia or irradiation for 6, 12, 24, 48 or 96 h. Flow cytometry was performed with a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) (Eisele et al., 2006). The following antibodies (R&D Systems) were used: anti-human CD62E, VEGF-R2 and VCAM-1. Signal intensity was calculated by dividing mean fluorescence obtained with specific antibody divided by signal intensity obtained with the isotype control antibody (specific fluorescence index, SFI).
Human tissue samples and tissue microarray
We investigated 120 glioblastoma samples (WHO grade IV) obtained from the tumour bank of the Institute of Neuropathology, University of Tuebingen derived from patients who underwent surgical treatment from 1993 to 2003 at the Department of Neurosurgery in Tuebingen or in the Department of Neurosurgery in Seesen. Diffuse astrocytomas WHO grade II (n = 10) and anaplastic astrocytomas WHO grade III (n = 10) were also investigated. All samples from diffuse and anaplastic astrocytomas were taken from primary tumours prior to therapy. Among 120 glioblastoma samples, 88 were derived from primary and 32 from recurrent tumours. Shortly after surgical removal, all specimens were fixed with 4% formalin (pH 7.4) and subsequently embedded in paraffin followed by preparation as tissue microarray (Warth et al., 2007). Further, 60 samples from 33 autopsy cases of a normal brain bank were added. Clinical data are summarized in Table 1. The tumour and normal brain tissues were stained with H&E, mouse anti-human antibodies to CD31 (Clone JC70A, Dako Cytomation, Glostrup, Denmark, 51 mg/ml), goat anti-human CD62E antibodies (Clone BBA18, R&D Systems, diluted 1:50) or the respective isotype control antibodies.
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Animal studies
On day 0, 104 SMA-560 cells were stereotactically implanted into the right striatum of VM/Dk mice. On day 7, LSK were isolated from the bone marrow of VM/Dk mice as described (Tabatabai et al., 2005
). Briefly, femora and tibiae of VM/Dk mice were flushed with 2% FCS-containing PBS, erythrocytes were lysed, bone marrow cells were incubated for 60 min at 4°C with the lineage marker antibodies rat anti-mouse CD4, CD8a, CD45R/B220, Gr-1, CD11b and TER119 (BD Biosciences Pharmingen, San Diego, CA, USA), then washed with PBS prior to the addition of pre-washed sheep anti-rat IgG magnetic beads (Dynabeads, Dynal, Biotech ASA, Oslo, Norway) at a 4 : 1 ratio of beads/cells. After 45 min, beads and attached cells were magnetically removed. The remaining lineage-depleted (lin–) cells were sorted with anti-Sca1+ beads by MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). The check of purity by flow cytometry revealed that <5% of the isolated LSK expressed CD4, CD8, CD11b, B220 or Gr-1. LSK were between 95% and 98% positive for Sca1 and >99% positive for c-kit (BD Biosciences Pharmingen). The LSK were cultured with murine IL-3 (20 ng/ml), IL-6 (50 ng/ml) and SCF (50 ng/ml) (R&D Systems) over night. On day 8, neutralizing rat anti-mouse CD62E (BD Bioscience, clone 10E9.6) or control antibodies were bolus-injected intraperitoneally (200 µl) and intravenously (5 µl). Four hours later, PKH26 (Sigma-Aldrich, Taufkirchen, Germany) stained LSK were injected intravenously. On day 10, mice were sacrificed. From each experimental group, three brains were analysed by flow cytometry and three brains by histology. For flow cytometry, 5 mm3 of the tumour-bearing as well as the contralateral hemisphere were dissociated in single cell suspension. For histology, 8 µm cryostat sections were stained with H&E and antibodies to vWF (Dako Cytomation) or CD62E (BD Biosciences). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Vectashield with DAPI, Vector Laboratories, Axxora GmbH, Loerrach, Germany).
Statistical analysis
Quantitative data were obtained for migration, ELISA, flow cytometry and reporter assays as indicated. Data are expressed as mean and SEM. Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test (Excel, Microsoft, Seattle, WA, USA). The experiments reported here were performed at least three times in triplicate with similar results.
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Results |
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Transendothelial HPC migration is enhanced after pretreatment of endothelial cells with SN-G
The molecular signature of endothelial cells depends on the environment. Endothelial cells from vessels within normal tissue are different from endothelial cells of vessels within inflamed or neoplastic tissue (Frenette et al., 1996b
). To simulate the conditioning of endothelial cells of vessels within glioblastoma tissue in vitro, we cultured cerebral endothelial cells (SV-HCEC) or HMEC in supernatant of LNT-229, LN-308, T113, T159 or T132 glioma cells. Further, we used BTEC from human glioblastoma specimens. HPC migrated through an untreated SV-HCEC-, HMEC (data not shown) and BTEC layer (Fig. 1). The migration was significantly enhanced after the pre-incubation of all three types of endothelial cell layer with different SN-G. Pretreatment of the SV-HCEC, HMEC (data not shown) and BTEC with hypoxia and irradiation also increased the migration of HPC. In contrast, incubation with supernatant of the astrocytic cell line SV-FHAS (SN-FHAS) did not modulate transendothelial migration. Thus, SN-G, hypoxia or irradiation alter the characteristics of endothelial cells and hence promote the transendothelial migration of HPC.
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SN-G induces the expression of CD62E on SV-HCEC and HMEC
To understand the increase in transendothelial HPC migration, we performed flow cytometry for the detection of VCAM-1 and CD62E on SV-HCEC, HMEC or BTEC either un- or pretreated with SN-G, irradiation or hypoxia. Untreated SV-HCEC, HMEC and BTEC constitutively expressed VCAM-1 at SFI values of 1.8, 1.7 and 1.9. The expression of VCAM-1 was not altered after pretreatment of the endothelial cells with SN-G, irradiation or hypoxia (data not shown). In contrast, CD62E was expressed on untreated BTEC but not on untreated SV-HCEC or HMEC. However, SN-G enhanced or induced the expression of CD62E as did hypoxia or irradiation (Fig. 2). Pretreatment with VEGF165 served as a positive control.
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Endothelial cells of tumour vessels in human astrocytomas but not normal brain vessels highly express CD62E
To confirm the relevance of CD62E induction on endothelial cells in vitro, we performed immunohistochemistry on human tissue microarray sampling normal brain sections, diffuse astrocytomas (WHO grade II), anaplastic astrocytomas (WHO grade III) and glioblastomas (WHO grade IV). Normal brain tissue contains small capillaries and vessels whereas the vessels in glioblastomas are characteristically composed of enlarged and thickened endothelial cell layers. CD31 staining labelled endothelial cells within the vessel walls of normal and neoplastic tissue. CD62E immunoreactivity was absent on endothelial cells of blood vessels within normal human brain samples. In contrast, CD62E was present in all neoplastic samples. CD62E strongly correlated with blood vessel morphology. Large vascular proliferations in glioblastoma samples showed the strongest CD62E immunoreactivity whereas smaller, more regular blood vessels found in the same tumours displayed a weaker CD62E staining intensity (Fig. 3). There was no difference between CD62E expression in samples derived from primary or recurrent glioblastomas. A slight cytoplasmic CD62E staining was detected in tumour cells of most tumour samples. This prompted us to determine the concentration of soluble CD62E (sCD62E) in SN-G and in supernatants of endothelial cells in vitro by ELISA. Supernatant of BTEC harvested after 96 h contained sCD62E (70 ± 10 pg/ml) whereas SN-G and supernatant of untreated SV-HCEC and HMEC did not. In contrast, 96 h after stimulating SV-HCEC and HMEC with SN-G, sCD62E was detected in the supernatant of these endothelial cells (50–70 pg/ml). Thus, CD62E shedding and release of sCD62E may account for the apparent cytoplasmic staining observed in astrocytoma samples (Fig. 3).
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Enhanced migration of HPC through an SV-HCEC layer in vitro depends on CD62E
We next investigated the role of HPC adhesion to CD62E for transendothelial HPC migration through an SN-G-conditioned endothelial cell layer. We studied the transendothelial migration in the absence or presence of neutralizing antibodies to CD62E either with or without pretreatment of the endothelial cell layer. Neutralizing CD62E reduced the transendothelial HPC migration significantly (Fig. 4A, first panel). Because of the important regulatory role of TGF-β for HPC glioma tropism (Tabatabai et al., 2006
), we also investigated the role of TGF-β in the transendothelial HPC migration by targeting TGF-β expression and TGF-β signalling in glioma cells. We used stably transfected LNT-229 siTGF-β1,2 cells (Friese et al., 2004; Eisele et al., 2006) or siRNA pools targeting TGF-β1 and TGF-β2, respectively, for transient transfection of LN-308 before collection of SN-G.
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Transendothelial HPC migration through cells that had been pretreated with supernatant of LNT-229 siTGF-β1,2 (SN-GsiTGF-β), was reduced and neutralizing
-CD62E did not modulate HPC migration (Fig. 4A, second panel). Further, transient transfection of LN-308 glioma cells with siRNA pools targeting TGF-β1 and TGF-β2 inhibited TGF-β1 and TGF-β2 release into the supernatant (Fig. 4B). Combined targeting of TGF-β1 and TGF-β2 in LN-308 cells inhibited the phosphorylation of Smad2 (Fig. 4B) indicating abrogation of TGF-β signalling. HPC migration through endothelial cells that had been pretreated with supernatant of these cells was accordingly reduced and neutralizing CD62E did not modulate the migration (Fig. 4A, third and fourth panel). Pre-irradiation of the endothelial cells resulted in an enhancement of transendothelial HPC migration (Fig. 1) that was also reduced by neutralizing CD62E antibodies (Fig. 4C). In contrast, neutralizing VCAM-1 antibodies did not significantly reduce HPC migration through an untreated SV-HCEC- or BTEC or an SN-G-treated SV-HCEC layer (Table 2). Exposure to hypoxia, irradiation, supernatant, neutralizing or control antibodies did not affect viability or proliferation of the endothelial cells or glioma cells (data not shown).
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Homing of murine HPC to orthotopically implanted SMA-560 gliomas in vivo is reduced after neutralization of CD62E
Subsequently, we investigated the role of CD62E for the glioma tropism of murine HPC in an orthotopic syngeneic glioma model in vivo. The 104 SMA-560 glioma cells were implanted into the right striatum of VM/Dk mice. On day 8, 106 PKH26-stained LSK cells were injected intravenously. Four hours before and 4 h after the injection of LSK, CD62E-neutralizing antibodies (six mice) or control antibodies (six mice) were bolus-injected both intraperitoneally and intratumourally in parallel in the same animals to block adhesion. On day 10, the brains were removed. Three brains per group were used for assessment by flow cytometry, three brains were analysed by histology. Injection of CD62E and control antibodies did not affect tumour morphology, tumour growth or vessel density as assessed by H&E or vWF staining (Fig. 5A). The endothelial cells of tumour vessels expressed CD62E (Fig. 5A). Fluorescence staining showed reduced numbers of PKH26-labelled cells within the SMA-560 gliomas after pretreatment with anti-CD62E antibodies (Fig. 5B). Single cell suspensions of freshly removed samples of the SMA-560 gliomas or of the contralateral hemisphere were analysed by flow cytometry to quantify the numbers of PKH26-labelled cells. Co-treatment with anti-CD62E antibody strongly reduced the amount of PKH26-positive LSK in orthotopic SMA-560 gliomas (Fig. 5C).
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TGF-β signalling in glioma cells is required for SN-G-mediated induction of CD62E expression on SV-HCEC
We then characterized the time course of CD62E induction in SV-HCEC and HMEC. TNF-
treatment (100 U/ml) served as a positive control. This treatment resulted in high expression of CD62E by SV-HCEC (Fig. 6A) and HMEC (Fig. 6B) after 6 h, then declining over the next 90 h. Similarly, treatment with VEGF165 (40 ng/ml) resulted in high CD62E expression after 6 h, which was no more detectable 96 h after treatment. Culturing of SV-HCEC or HMEC in supernatant of the astrocytic cell line SV-FHAS did not induce CD62E expression. Treatment of SV-HCEC or HMEC with SN-G resulted in a maximum CD62E expression at 12 h (Fig. 6A and B).
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Next, we analysed the role of TGF-β signalling for the induction of CD62E. The treatment of SV-HCEC with recombinant TGF-β2 (10 ng/ml) did not induce CD62E expression (Fig. 7A). We also collected supernatant of glioma cells that had been pretreated with the TGF-β receptor I kinase inhibitor, SD-208 (0.1 µmol/l and 1.0 µmol/l). SD-208 treatment blocked TGF-β signalling in LN-308, T113 and T132 cells (Fig. 7B). The exposure of SV-HCEC to supernatant of these SD-208-treated glioma cells did not induce CD62E expression (Fig. 7A). In accordance, exposure of SV-HCEC to SN-308 siTGF-β1,2 (Fig. 4A and B) did not induce CD62E (Fig. 7A). Finally, we also treated LNT-229 siTGF-β1,2 cells stably depleted from TGF-β1 and TGF-β2 (Friese et al., 2004
) with recombinant TGF-β2 (10 ng/ml) for 7 days to rescue TGF-β signalling in these glioma cells (Friese et al., 2004; Tabatabai et al., 2005; Eisele et al., 2006). Supernatant from these cells again induced CD62E on SV-HCEC (Fig. 7A). In contrast, the treatment of SV-HCEC with SN-G in the presence of SD-208, which abrogates TGF-β signalling pathway in SV-HCEC and HMEC (Fig. 7C), did not influence the SN-G-mediated induction of CD62E. The exposure of endothelial cells to SN-GRT or SN-GHO led to the induction of CD62E, too. This effect was again prevented by SD-208 treatment of the glioma cells before harvesting the supernatant (data not shown).
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The experiments involving the addition of recombinant TGF-β2 or SD-208 indicate that (i) TGF-β signalling in glioma cells is required for the induction of CD62E on endothelial cells; but (ii) TGF-β signalling in SV-HCEC is not involved in SN-G-mediated induction of CD62E expression.
Induced CD62E expression on SV-HCEC is mediated by VEGF-R2 signalling
We next analysed the levels of VEGF, a candidate inducer of CD62E expression, in concentrated SN-Gp and SN-GsiTGF-β from cells treated with or without SD-208 or TGF-β2 (Table 3). VEGF release was reduced by abrogated TGF-β signalling. In contrast, addition of recombinant TGF-β2 to LNT-229 siTGF-β1,2 cells for 7 days before generation of supernatant restored VEGF levels in the supernatant. Further, irradiated and hypoxic SV-HCEC released increased levels of VEGF (Table 3). SV-HCEC, HMEC and BTEC expressed the corresponding receptor VEGF-R2 (Fig. 8A). Therefore, we analysed the role of VEGF/VEG-R2 signalling in endothelial cells for CD62E induction. Treatment of SV-HCEC, HMEC or BTEC with VEGF165, SN-G, hypoxia or irradiation increased the levels of phosphorylated VEGF-R2. NIH3T3 cells were used as negative controls (Fig. 8B). The levels of total VEGF-R2 in untreated SV-HCEC (597 pg/ml) did not increase by exposure of SV-HCEC to VEGF165 (590 pg/ml) or supernatant of LN-308 (572 pg/ml). Pretreatment with hypoxia, 2 Gy or 8 Gy, however, increased the levels of total VEGF-R2 in SV-HCEC (807, 992 and 2670 pg/ml). Similarly, the levels of total VEGF-R2 in untreated HMEC (664 pg/ml) was not increased by exposure to VEGF165 (579 pg/ml), supernatant of LN-308 (660 pg/ml), whereas hypoxia (772 pg/ml), irradiation at 2 Gy (1222 pg/ml) or 8 Gy (2982 pg/ml) led to elevated levels of total VEGF-R2 protein in HMEC. In BTEC, we detected levels of total VEGF-R2 in untreated (502 pg/ml), hypoxic (452 pg/ml) or irradiated cells (2 Gy: 459, 8 Gy 449 pg/ml). These data indicate that VEGF-R2 signalling activity in SV-HCEC, HMEC and BTEC is enhanced in response to SN-G, hypoxia or irradiation. Targeting of VEGF in glioma cells by siRNA pools led to a reduced VEGF release by LN-308 glioma cells or T113 primary cultures (Fig. 8C). VEGF levels were also are reduced in supernatant of LN-308 siTGF-β1,2 (Fig. 8C) and supernatant of LNT-229 after inhibition of TGF-β signalling (Table 3). The SN-G-mediated de novo expression of CD62E was prevented by VEGF gene silencing and by neutralizing VEGF or VEGF-R2 antibodies (Fig. 8D). Further, the exposure of SV-HCEC to irradiation or SN-G, SN-GRT or SN-GHO supplemented with neutralizing -VEGF and -VEGF-R2 antibodies prevented CD62E induction (data not shown). Thus, (i) SN-G enhances VEGF-R2 signalling activity in endothelial cells; (ii) VEGF from SN-G, SN-GHO or SN-GRT is required for the SN-G-induced expression of CD62E in SV-HCEC; (iii) VEGF release from SV-HCEC is required for CD62E induction after exposure to hypoxia or irradiation.
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Transcriptional activation of CD62E in endothelial cells is mediated by NF
BThe promoter of CD62E contains multiple NF
B-binding sites (Schindler et al., 1994). We detected NFB p65 and p50 in whole cell lysates of SV-HCEC (Fig. 9A). Protein levels were not modulated by irradiation, hypoxia or exposure to SN-Gp, SN-GsiTGF-β or SN-GRT. Futher, we analysed the activation of the NFB transcription family in nuclear extracts of BTEC, SV-HCEC and HMEC (Fig. 9B). Raji extracts served as positive control, nuclear extracts from NIH3T3 cells were used as negative controls. The NFB transcription factor family is activated in untreated endothelial cells. We next analysed the activity of p50 in nuclear extracts of endothelial cells after treatment with VEGF165 or SN-G. TNF- served as positive control (Fig. 9C). VEGF165 and SN-G further increased the transcriptional activity of p50 whereas treatment with supernatant of LN-308 siVEGF did not. In addition, we performed reporter assays with pNFB-luc. This plasmid contains four copies of the NFB consensus sequence and detects the transcriptional activity of NFB. Treatment with TNF- (100 U/ml) served as a positive control (Figs 9 and 10). The relative luciferase activity of pNFB-luc in untreated BTEC, SV-HCEC and HMEC demonstrates the baseline transcriptional activity (Fig. 9D) in accordance with the data of the transAM assay (Fig. 9B and C). After exposure to TNF-, VEGF165 or SN-G, NFB transcriptional activity significantly increased. Neutralizing antibodies to VEGF and VEGF-R2 reduced SN-G-induced NFB transcriptional activity (Fig. 10).
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In parallel, we analysed the activity of the CD62E promoter by measuring the relative luciferase activity of pGL3basic.CD62E. In this plasmid, the reporter gene is driven by the CD62E promoter and indicates the activity of the CD62E promoter. We detected relative luciferase activity in untreated BTEC indicating a baseline promoter activity in accordance with the data from flow cytometry (Fig. 2). In contrast, luciferase activity was absent in untreated SV-HCEC or HMEC, indicating a lack of CD62E promoter activity in this cell population (Fig. 9D). After treatment with TNF-
, VEGF165 or SN-G, however, the relative luciferase activity of pGL3.basic.CD62E increased (Fig. 10). Irradiation with 2 Gy, 8 Gy or hypoxia also increased pGL3.basic.CD62E activity (data not shown). NFB transcriptional activity and CD62E promoter activity were reduced by neutralizing -VEGF and -VEGF-R2 antibodies (Fig. 10). To analyse whether NFB transcriptional activity is required for CD62E promoter activity, we performed reporter assays after targeting NFB by the NFB-super-repressor Ad-IKBM. We transduced SV-HCEC with 100 MOI of Ad-IKBM. Ad-Mock served as control. Relative luciferase activity of pGL3.basic.CD62E in SV-HCEC was not induced by SN-G in the presence of the NFB super repressor (Fig. 10). These experiments place VEGF signalling and NFB upstream of the transcriptional activation of CD62E in SV-HCEC. The presumptive signalling cascade leading to the induction of CD62E in SV-HCEC by SN-G, irradiation or hypoxia is summarized in Fig. 11.
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Adult HPC migrate towards experimental gliomas. Therefore, this adult stem cell population might be a promising cellular vector for the delivery of therapeutic molecules to experimental gliomas. Before applying therapeutic approaches, however, the underlying molecular mechanisms of the glioma-mediated HPC attraction should be better understood. The objective in characterizing the glioma tropism of HPC is to achieve an optimal seeding of experimental gliomas with HPC. Adhesion molecules expressed on endothelial cells serve as keys to allow the entrance of circulating cells at specific tissue sites (Frenette et al., 1996a
). Bone marrow- and umbilical cord blood-derived CD34+ HPC adhere to CD62E on bone marrow microvasculature (Najyer et al., 1999; Dimitroff et al., 2001; Hidalgo et al., 2002; Greenberg et al., 2000). P- and E-selectin-deficient mice exhibit severe deficiencies in haematopoiesis (Frenette et al., 1996). Intravital microscopy studies show that P- and E-selectin contribute to HPC rolling in the bone marrow microvasculature. Blocking both selectins and VCAM-1 inhibits the rolling interaction almost completely (Mazo et al., 1998). Hidalgo et al. (2002) demonstrated by fluorescence intravital microscopy and homing assays in NOD/SCID mice that endothelial selectins are necessary for CD34+ HPC homing: rolling on bone marrow endothelium and retention in the bone marrow compartment are severely reduced in CD62E-deficient NOD/SCID mice. Thus, homing of HPC to the bone marrow is mediated by HPC interactions with multiple adhesion receptors. Among these, CD62E and VCAM-1 are expressed preferentially on endothelial cells. Therefore, we focused on investigating the possible contribution of CD62E and VCAM-1 to HPC glioma tropism.
CD62E expression in glioma-conditioned endothelial cells in vitro and in vivo
We used three different endothelial cell types: the cerebral endothelial cell line SV-HCEC (Muruganandam et al., 1997), the endothelial cell line HMEC and BTEC (Miebach et al., 2006). These endothelial cells constitutively express VCAM-1, but not CD62E. In the bone marrow microvasculature, CD62E and VCAM-1 are constitutively expressed (Schweitzer et al., 1996). In most other tissues, however, CD62E is transcriptionally regulated and expressed on the surface after exposure to specific inflammatory stimuli (Bevilacqua et al., 1989). We found CD62E expression on untreated BTEC. De novo CD62E expression by SV-HCEC and HMEC was induced after stimulation with SN-G, SN-GHO or SN-GRT, as well as treatment with hypoxia (Fig. 2) or irradiation. The constitutive VCAM-1 expression on the endothelial cells was not altered by these treatments. These results differ from studies with bone marrow microvasculature: total body irradiation of mice increased VCAM-1 expression, but the constitutive expression level of CD62E within the bone marrow microvasculature was unchanged (Mazo et al., 2002). Having observed an SN-G-induced CD62E expression by SV-HCEC and HMEC in vitro, we analysed CD62E expression in situ. The endothelium of vessels within astrocytoma tissue of WHO grades II–IV highly expressed CD62E whereas CD62E staining was absent in normal brain (Fig. 3). In addition to the well-defined CD62E staining of tumour endothelium, there was a diffuse extracellular and cytoplasmic staining in the neoplastic specimens. This might indicate the shedding of CD62E and the presence of the soluble form of CD62E (Oh et al., 2007). In that regard, we also detected sCD62E in supernatant of stimulated SV-HCEC, HMEC and in supernatant of untreated BTEC in vitro.
CD62E is one mediator of the glioma-mediated HPC attraction in vitro and in vivo
The transendothelial migration of HPC was significantly diminished in the presence of neutralizing CD62E antibodies (Fig. 4) whereas neutralizing VCAM-1 antibodies had no such effect. In vivo, administration of a CD62E-blocking antibody prevented the accumulation of PKH26-positive LSK in orthotopic SMA-560 gliomas (Fig. 5), indicating that CD62E is required for the glioma tropism of LSK in vivo. In a model of chronic kidney disease, Gong et al. (2006) demonstrated de novo CD62E expression in renal vascular endothelium mediating leucocyte adhesion. The bolus injection of neutralizing CD62E antibodies diminished interstitial inflammation and macrophage sequestration in the kidney in vivo. Recently, Nishiwaki et al. (2007) demonstrated a crucial role for CD62E in the interaction between circulating endothelial progenitor cells and vessel endothelium in the mouse hind-limb ischaemia paradigm. Oh and colleagues (2007) demonstrated that CD62E is a pivotal molecule for endothelial progenitor cell homing to the ischaemic site and for vasculogenesis in an ischaemic limb model. Based on these data, a novel cell-based therapy for ischaemic atherosclerosis might be designed.
SN-G-mediated induction of CD62E requires TGF-β signalling in glioma cells and VEGF/VEGF-R2 signalling in endothelial cells
We next investigated the molecular mechanism mediating the de novo expression of CD62E in SV-HCEC or HMEC. Culturing of SV-HCEC with SN-GsiTGF-β did not induce CD62E expression (Fig. 7). HPC migration through an SN-GsiTGF-β-treated SV-HCEC layer was not altered by neutralizing -CD62E. The reduction of relative HPC migration (Fig. 4A) observed after pretreating SV-HCEC with SN-GsiTGF-β can be explained by the lack of CD62E expression (Fig. 7). Based on this observation, we analysed the role of TGF-β signalling in the glioma-mediated induction of CD62E. When TGF-β signalling was inhibited in LNT-229 glioma cells before supernatant collection—either by SD-208 or by siRNA—these supernatants did not induce CD62E expression (Fig. 7). Inhibition of TGF-β signalling in SV-HCEC or HMEC by SD-208, however, did not modulate SN-G-mediated CD62E induction. Further, treatment of SV-HCEC with recombinant TGF-β2 alone did not induce CD62E (Fig. 7). We concluded that glioma-mediated CD62E induction requires intact TGF-β signalling in the glioma cells, but not in SV-HCEC. Inhibition of TGF-β signalling in glioma cells also led to diminished VEGF levels (Table 3, Fig. 8C). Irradiation with 2 or 8 Gy and pre-exposure to hypoxia on the other hand increased VEGF secretion by SV-HCEC and HMEC (Table 3). We therefore analysed the influence of VEGF/VEGF-R2-signalling on CD62E induction in SV-HCEC. Treatment of SV-HCEC and HMEC with supernatant of glioma cells that had been transfected with siRNA pools targeting VEGF did not induce CD62E (Fig. 8D). Neutralization of VEGF or VEGF-R2 bioactivity impaired SN-G-mediated CD62E induction (Fig. 8D) indicating a pivotal role of this signalling pathway in SV-HCEC or HMEC in the induction of CD62E expression. Recently, Stannard et al. (2007) demonstrated that VEGF primes endothelial cells by sensitizing them to cytokines, leading to enhanced selective pro-inflammatory responses, including upregulation of CD62E.
De novo expression of CD62E by endothelial cells is mediated by NFB
To further characterize the downstream events after VEGF-R2 activation, we performed reporter assays. SN-G, irradiation or hypoxia induced CD62E promoter activity in SV-HCEC. Since the CD62E promoter contains three NFB binding sites (Schindler et al., 1994), we analysed the role of NFB in our paradigm. NFB is expressed and transcriptionally active in SV-HCEC, HMEC and BTEC (Figs 9 and 10). NFB is also activated in pre-sheared HUVEC leading to NFB-dependent cytoprotective responsiveness (Patridge et al., 2007). Consequently, we investigated the upstream control of CD62E promoter activity with the NFB superrepressor, Ad-IkBM. Inhibition of NFB acitivity strongly reduced CD62E promoter activity (Fig. 10). This central role of NFB activity for CD62E induction in our studies parallels results in other models: endothelial cell activation by HIV infection results in CD62E expression via a Tat-induced NFB-mediated pathway (Cota-Gomez et al., 2002). CD62E induction within an atherosclerotic lesion is evoked by the platelet-specific chemokine 4 derived from platelets and activation of NFB was crucial for CD62E induction on endothelium by platelet-specific chemokine 4 (Yu et al., 2005).
In summary, we define a signalling cascade leading to CD62E induction on endothelial cells in response to treatment with SN-G, SN-GHO, SN-GRT, irradiation or hypoxia. This cascade involves a TGF-β-dependent release of VEGF by glioma cells and the subsequent activation of VEGF-R2 in endothelial cells resulting in NFB-mediated CD62E expression. We observed a selective expression of this adhesion molecule in human astrocytoma samples of WHO grades II–IV and demonstrated the functional relevance of CD62E for transendothelial HPC migration in vitro and in vivo. Therefore, we conclude that adhesion to CD62E on tumour endothelium might be a pivotal first step in the glioma tropism of HPC. Given the central role of VEGF in the glioma-mediated induction of CD62E on endothelial cells, a putative HPC-based therapy against glioblastomas might not be effective in combination with antiangiogenic strategies targeting VEGF, e.g. enzastaurin or bevacizumab.
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Acknowledgements |
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This work was supported by the Landesstiftung Baden-Württemberg, State of Baden-Württemberg, Germany (P-LS-AS/HSPA7-12) to W.W. and by the Deutsche Forschungsgemeinschaft (We 1502/10-4) to M.W. and W.W.
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