The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines
2001; Springer Nature; Volume: 20; Issue: 20 Linguagem: Inglês
10.1093/emboj/20.20.5568
ISSN1460-2075
Autores Tópico(s)Protease and Inhibitor Mechanisms
ResumoArticle15 October 2001free access The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines Eric Guenzi Eric Guenzi Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Kristin Töpolt Kristin Töpolt Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Emmanuelle Cornali Emmanuelle Cornali Department of Virus Research, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany Present address: Ingenium Pharmaceuticals AG, Frauenhoferstrasse 13, D-82152 Martinsried, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Clara Lubeseder-Martellato Clara Lubeseder-Martellato Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Anita Jörg Anita Jörg Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Kathrin Matzen Kathrin Matzen Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Christian Zietz Christian Zietz Institute of Pathology, Ludwig Maximilians University, D-80337 Munich, Germany Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, D-81377 Munich, Germany Search for more papers by this author Filomena Nappi Filomena Nappi Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Martin Schwemmle Martin Schwemmle Department of Virology, Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany Search for more papers by this author Christine Hohenadl Christine Hohenadl Present address: Austrian Nordic Biotherapeutics, Veterinärplatz 1, 1210 Vienna, Austria Search for more papers by this author Giovanni Barillari Giovanni Barillari Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Erwin Tschachler Erwin Tschachler Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Paolo Monini Paolo Monini Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Barbara Ensoli Barbara Ensoli Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Michael Stürzl Corresponding Author Michael Stürzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Eric Guenzi Eric Guenzi Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Kristin Töpolt Kristin Töpolt Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Emmanuelle Cornali Emmanuelle Cornali Department of Virus Research, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany Present address: Ingenium Pharmaceuticals AG, Frauenhoferstrasse 13, D-82152 Martinsried, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Clara Lubeseder-Martellato Clara Lubeseder-Martellato Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Anita Jörg Anita Jörg Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Kathrin Matzen Kathrin Matzen Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Christian Zietz Christian Zietz Institute of Pathology, Ludwig Maximilians University, D-80337 Munich, Germany Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, D-81377 Munich, Germany Search for more papers by this author Filomena Nappi Filomena Nappi Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Martin Schwemmle Martin Schwemmle Department of Virology, Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany Search for more papers by this author Christine Hohenadl Christine Hohenadl Present address: Austrian Nordic Biotherapeutics, Veterinärplatz 1, 1210 Vienna, Austria Search for more papers by this author Giovanni Barillari Giovanni Barillari Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Erwin Tschachler Erwin Tschachler Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria Search for more papers by this author Paolo Monini Paolo Monini Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Barbara Ensoli Barbara Ensoli Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy Search for more papers by this author Michael Stürzl Corresponding Author Michael Stürzl Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany Search for more papers by this author Author Information Eric Guenzi1,9, Kristin Töpolt1,9, Emmanuelle Cornali2,3,9, Clara Lubeseder-Martellato1,9, Anita Jörg1, Kathrin Matzen1, Christian Zietz4, Elisabeth Kremmer5, Filomena Nappi6, Martin Schwemmle7, Christine Hohenadl8, Giovanni Barillari6, Erwin Tschachler9, Paolo Monini6, Barbara Ensoli6 and Michael Stürzl 1 1Department of Virus-induced Vasculopathy, Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany 2Department of Virus Research, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany 3Present address: Ingenium Pharmaceuticals AG, Frauenhoferstrasse 13, D-82152 Martinsried, Germany 4Institute of Pathology, Ludwig Maximilians University, D-80337 Munich, Germany 5Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, D-81377 Munich, Germany 6Laboratory of Virology, Retrovirus Division, Istituto Superiore di Sanità, 00161 Rome, Italy 7Department of Virology, Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany 8Present address: Austrian Nordic Biotherapeutics, Veterinärplatz 1, 1210 Vienna, Austria 9Department of Dermatology, Division of Immunology, Allergy, and Infectious Diseases, University of Vienna Medical School, 1090 Vienna, Austria ‡E.Guenzi, K.Töpolt, E.Cornali and C.Lubeseder-Martellato contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5568-5577https://doi.org/10.1093/emboj/20.20.5568 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases. Introduction Activation of endothelial cells is implicated in numerous physiological and pathological processes including cell-mediated immunity and angiogenesis during development, inflammation or tumor growth (reviewed by Cines et al., 1998; Carmeliet and Jain, 2000). The activated endothelial cell phenotype represents a time- and dose-integrated response to various stimuli originating from the blood and/or from neighboring cells and tissues. Inflammatory cytokines (IC) such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as angiogenic growth factors (AGF) such as vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF), are the best characterized and most potent paracrine regulators of endothelial cell activity. For example, IC activate endothelial cell adhesiveness for leukocytes (Bevilacqua et al., 1985; Cavender et al., 1991) and are potent inhibitors of endothelial cell proliferation (Frater-Schroder et al., 1987; Friesel et al., 1987; Schweigerer et al., 1987; Cozzolino et al., 1990). In contrast, VEGF and bFGF activate endothelial cell proliferation (Folkman and Klagsbrun, 1987; Ferrara and Henzel, 1989; Keck et al., 1989). VEGF, in addition, induces endothelial cell adhesiveness for leukocytes (Melder et al., 1996; Detmar et al., 1998), whereas bFGF inhibits the expression of adhesion molecules on endothelial cells (Griffioen et al., 1996a,b). In vivo, several of these factors are simultaneously present and active on endothelial cells. Moreover, the effect of a single factor on the endothelium may vary from tissue to tissue. For example, IC have been shown to induce angiogenesis in some in vivo models (Frater-Schroder et al., 1987; Mahadevan et al., 1989; Montrucchio et al., 1994; Gerol et al., 1998; Torisu et al., 2000) or to inhibit angiogenesis in others (Cozzolino et al., 1990; Norioka et al., 1994; Yilmaz et al., 1998; Fathallah-Shaykh et al., 2000). In addition, according to their concentrations IC may act either as pro- or anti-angiogenic molecules in the same model systems (Fajardo et al., 1992). The angiogenic effect of IC has been attributed to the recruitment of monocytes into tissues that, in turn, may release angiogenic factors (Frater-Schroder et al., 1987; Fajardo et al., 1992; Montrucchio et al., 1994; Joseph and Isaacs, 1998) or to the induction of bFGF or VEGF expression in resident cells (Samaniego et al., 1997; Torisu et al., 2000). The anti-angiogenic effect of IC may be caused by their anti-proliferative activity. However, only little is known about how IC can induce opposite effects on angiogenesis. The aim of this study was to identify genes that mediate the anti-proliferative effect of IC on endothelial cells and that may characterize the IC-activated endothelial cell phenotype in tissues. To this goal, gene expression of endothelial cells in the presence of IC or AGF was analyzed by differential display RT–PCR (DDRT–PCR) (Liang and Pardee, 1992). Using this approach, we detected the human guanylate binding protein-1 (GBP-1), which belongs to the group of large GTP-binding proteins (Prakash et al., 2000a). GBP-1 was found to be strongly induced by IC and downregulated by AGF. Detailed analyses of GBP-1 expression in cultivated microvascular and macrovascular endothelial cells and in vessel endothelial cells of Kaposi's sarcoma (KS) tissues demonstrated that GBP-1 characterizes the IC-activated non-proliferating phenotype of endothelial cells in vitro and in vivo. Additionally, we showed that GBP-1 mediates the anti-proliferative effect of IC on endothelial cells without affecting cell adhesiveness. The anti-proliferative activity of GBP-1 was found to be independent of its GTPase function, but was found to be mediated by the C-terminal helical domain of the molecule. Results GBP-1 expression is induced in endothelial cells in vitro by IL-1β, TNF-α and IFN-γ Initial studies were carried out to determine the appropriate concentrations of IC (IL-1β, TNF-α, IFN-γ) or AGF (bFGF, VEGF) that clearly affected proliferation of both human macrovascular umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MVEC). Combined application of bFGF and VEGF in concentrations of 10 ng/ml each potently activated endothelial cell proliferation of both cell types, whereas IL-1β, IFN-γ and TNF-α inhibited this AGF-induced proliferation in a dose-dependent manner (Figure 1A). In order to identify genes that may mediate the anti-proliferative effect of IC on endothelial cells and may characterize the IC-activated endothelial cell phenotype, the pattern of endothelial cell gene expression in the presence of IC or AGF was compared by DDRT–PCR in MVEC (data not shown). Figure 1.Inflammatory cytokines inhibit endothelial cell proliferation and induce expression of GBP-1. (A) Proliferation assay with HUVEC and MVEC upon addition of AGF (VEGF and bFGF, 10 ng/ml each, except control) and increasing concentrations of IL-1β, TNF-α or INF-γ. (B) Northern blot (upper panel) and western blot (lower panels) analyses of GBP-1 expression in MVEC incubated with either bFGF (10 ng/ml), VEGF (10 ng/ml), IL-1β (20 U/ml), TNF-α (300 U/ml), IFN-γ (100 U/ml), or control medium for 5 h and 8 h, respectively. Polyclonal rabbit anti-GBP-1 peptide antibody (αGBP-1 pAb), polyclonal rabbit anti-GBP-1 antibody (αGBP-1 rAb), actin control (Actin). (C) Immunostaining for detection of GBP-1 expression in HUVEC stimulated for 8 h as described in (B). (D) Northern blot analyses of GBP-1 mRNA expression in MVEC after incubation for various periods of time with IL-1β; (E) in the presence or absence of cycloheximide (CHX, 50 μM) and IL-1β (20 U/ml, 5 h induction). (F) RT–PCR analysis of IFN-γ expression in HUVEC and HUT 78 lymphocytes stimulated for 5 h with IL-1β (20 U/ml) and phytohemagglutinin-L (PHA, 5 μg/ml), respectively. Actin mRNA was amplified as a control. Download figure Download PowerPoint The gene encoding GBP-1 was the only one of several differently expressed genes identified in the DDRT–PCR study that was upregulated by IL-1β, TNF-α or IFN-γ at both the mRNA (Figure 1B, upper panel) and protein (Figure 1B, lower panels) levels. In contrast, VEGF- or bFGF-treated or untreated cells only weakly expressed GBP-1 (Figure 1B). These results were confirmed at the single cell level by immunocytochemistry (Figure 1C). IL-1β-induced GBP-1 mRNA expression reached the maximal level at 5 h after stimulation, remaining high for at least 24 h (Figure 1D). This induction was not secondarily mediated via induction of IFN-γ expression because it was also observed when cellular protein synthesis was abolished by cycloheximide (Figure 1E). In addition, IL-1β did not induce IFN-γ expression in HUVEC (Figure 1F). GBP-1 mRNA and protein expression induced by IC correlate inversely with endothelial cell proliferation in vitro Experiments with combined IC and AGF showed that GBP-1 expression can be induced by IL-1β, TNF-α or IFN-γ in the presence of AGF either by adding the cytokines to the cells simultaneously (Figure 2A) or in AGF-pretreated cells (Figure 2C). On the other hand, IC-induced GBP-1 expression was consistently reduced by the addition of AGF, both in HUVEC and MVEC (Figure 2B). A more detailed analysis confirmed that VEGF as well as bFGF significantly reduced IL-1β-induced GBP-1 expression and that both factors together had additive inhibitory effects on GBP-1 expression at both the RNA (Figure 2D, white columns) and protein (Figure 2E, white columns) levels. Most importantly, in the presence of IC endothelial cell proliferation (Figure 2D and E, black columns) was inversely related to GBP-1 mRNA and protein expression (Figure 2D and E, white columns; compare also Figures 1A and 2A). This indicated that GBP-1 characterizes the IC-activated non-proliferating phenotype of endothelial cells in vitro. Figure 2.IC-induced GBP-1 expression is inversely related to endothelial cell proliferation. (A) Western blot analyses of GBP-1 expression in HUVEC and MVEC treated as described in Figure 1(A), (B) after the addition of IL-1β (200 U/ml), TNF-α (300 U/ml) and IFN-γ (100 U/ml) alone or in the presence of AGF (VEGF and bFGF, 10 ng/ml each; corresponding signal intensities were densitometrically determined, lower panels) and (C) after the addition of IL-1β (20 U/ml), TNF-α (300 U/ml) or IFN-γ (100 U/ml) to HUVEC that were pre-incubated overnight (O.N.) in the presence or absence of AGF. (D) Northern blot and (E) western blot analyses of GBP-1 expression in MVEC after the addition of AGF or IL-1β (20 U/ml), alone or combined. Recombinant His6-tagged GBP-1 was used as a protein standard for quantification. Corresponding signal intensities were densitometrically determined (lower panels, white columns) and compared with the proliferative capacity of MVEC under the same conditions (lower panels, black columns). Ethidium bromide (EtBr) visualization of blotted RNA, actin control (Actin). Northern blot analyses were carried out 5 h and western blot analyses 24 h after stimulation of the cells. A monoclonal rat anti-GBP-1 antibody was used for the detection of GBP-1. Download figure Download PowerPoint GBP-1 expression correlates inversely with endothelial cell proliferation in KS in vivo To investigate whether GBP-1 expression may define the IC-induced non-proliferating phenotype of endothelial cells in vivo also, double staining experiments and in situ hybridization studies were performed on KS (reviewed by Ensoli et al., 2001; Stürzl et al., 2001). KS lesions were chosen as the in vivo model because they are highly vascularized (Figure 3A, black arrows) due to the production of bFGF and VEGF by the KS spindle cells, which are regarded as the tumor cells of KS [Figure 3A, white arrows (Xerri et al., 1991; Ensoli et al., 1994; Cornali et al., 1996)]. However, lesions are also infiltrated by monocytes and lymphocytes, which locally produce IL-1β (Figure 3B, arrows), TNF-α (Figure 3C, arrows) and IFN-γ (Figure 3D, arrows), which activates endothelial cells (Stürzl et al., 1995; Fiorelli et al., 1998). Figure 3.GBP-1 is expressed in vessel endothelial cells of KS. (A–D) Staining of KS tissue sections by hematoxylin–eosin [A, blood vessels (black arrows), KS spindle cells (white arrows)] and by immunohistochemistry for detection of IL-1β (B, arrow), TNF-α (C, arrow) and IFN-γ (D, arrow). (E) Simultaneous staining of GBP-1 (brown, arrow) and the monocytic marker protein CD68 (red). (F–H) Immunodetection of GBP-1 in endothelial cells (F, arrow) and control staining of consecutive sections in the absence of the anti-GBP-1 antibody (G) or in the presence of a 320-fold molar excess of purified recombinant GBP-1 protein (H). (I–L) In situ hybridization for detection of GBP-1 RNA (I, bright field; J, corresponding dark field). Overview pictures of control hybridizations with the GBP-1 sense control probe (K, bright field; L, dark field). Download figure Download PowerPoint Tissue sections were stained simultaneously for GBP-1 protein and the monocyte specific marker CD68 (Figure 3E). GBP-1 was selectively detected in endothelial cells of vessels (Figure 3E, brown, arrow) that are surrounded by numerous perivascular monocytes (Figure 3E, red). Consecutive control sections of a positive specimen with GBP-1-expressing endothelial cells (Figure 3F, arrow) did not show any staining when the primary anti-GBP-1 antibody was omitted (Figure 3G) or applied in the presence of an excess of the purified recombinant GBP-1 protein (Figure 3H). GBP-1 expression in vessel endothelial cells was confirmed at the RNA level by in situ hybridization using a GBP-1-specific antisense RNA probe (Figure 3I, bright field; J, corresponding dark field, arrow). Control hybridizations with the GBP-1 sense probe did not produce any signals (Figures 3K and L). To investigate whether GBP-1 expression may define the non-proliferating phenotype of vessel endothelial cells in KS, immunofluorescence studies for the simultaneous detection of GBP-1, the endothelial cell-associated antigen CD31 or the proliferation-associated antigen Ki67 were performed. Simultaneous expression of GBP-1 (Figure 4A, arrow; C, green) and CD31 (Figure 4B, arrow; C, red) was found only in endothelial cells of some well-differentiated vessels (Figure 4C, GBP-1/CD31 colocalization, yellow). No staining was observed in the epidermal layer overlaying KS (Figure 4C, asterisk) and in the perivascular areas containing the KS spindle cells, monocytes, lymphocytes, fibrocytes and smooth muscle cells (cellular composition of KS reviewed by Stürzl et al., 2001). In contrast, in no case were GBP-1 (Figure 4D and G, arrows; F and I, green) and Ki67 (Figure 4E and H, arrows; F and I, red) found to be co-expressed in the same cell (Figure 4F and I), demonstrating that GBP-1 is not expressed in proliferating endothelial cells. Specificity of Ki67 staining was demonstrated by the positive reaction of proliferating basal cells in the epidermis (Figure 4I, asterisk). Figure 4.GBP-1 expression correlates inversely with endothelial cell proliferation in KS. (A–C) Immunofluorescence staining of KS tissue sections for the detection of GBP-1 and CD31 antigens. Detection of GBP-1 (A, arrow), CD31 (B, arrow) alone and in combination (C, GBP-1, green cytoplasmic staining; CD31, red cytoplasmic staining, colocalization yellow). The epidermal layer overlaying the lesions is indicated by an asterisk. (D–I) Detection of GBP-1 (D and G, arrows) and Ki67 (E and H, arrows) alone and in combination (F and I, GBP-1, green, Ki67, red nuclear staining) in different KS specimens. Ki67 positive basal cells in the epidermis are labeled by an asterisk. (J–M) Triple labeling experiment for the detection of GBP-1 (J, green arrow), Ki67 (K, blue arrow) and CD31 (L, red arrow) alone or in combination (M, CD31, red; GBP-1, green; Ki67, blue). Ki67/CD31 (blue arrows) and GBP-1/CD31 (yellow arrows) double-positive endothelial cells are indicated. Download figure Download PowerPoint To further prove that GBP-1 is only expressed in non-proliferating but not in proliferating endothelial cells within KS lesions, triple labeling experiments for the simultaneous detection of GBP-1, Ki67 and CD31 were performed. CD31-positive endothelial cells surrounding tumor vessels were evenly distributed in the tissue sections (Figure 4L, arrow and M, red cytoplasmic staining). In contrast, the highest numbers of GBP-1- (Figure 4J, arrow and M, green cytoplasmic staining) or Ki67- (Figure 4K, arrow and M, blue nuclear staining) positive endothelial cells were detected in different areas of the tissue section. Interestingly, areas with many Ki67-expressing endothelial cells (Figure 4M, upper part, blue arrows) revealed only few GBP-1-expressing endothelial cells, and vice versa, in areas with many GBP-1-expressing endothelial cells (Figure 4M, lower part, yellow arrows) only a few Ki67 positively stained cells were detected. The opposite local distribution of GBP-1- or Ki67-expressing endothelial cells in KS tissue sections was also observed when GBP-1 expression was investigated at the RNA level by in situ hybridization (data not shown). Altogether, these data demonstrate that in KS tissues GBP-1 expression is closely associated with vessel endothelial cells and specifically detected in areas with high IC expression, whereas its expression is low or absent in proliferating endothelial cells. Since GBP-1 expression both in vitro and in vivo was inversely related to endothelial cell proliferation these data strongly suggest that this protein may mediate the anti-proliferative effect of IC on these cells. GBP-1 mediates the IC-induced inhibition of endothelial cell proliferation To determine whether GBP-1 mediates the anti-proliferative effect of IC, HUVEC were transduced with retroviral vectors that express a GBP-1 full-length cDNA in sense (Figure 5A, S) or antisense (Figure 5A, AS) orientation or with the control vector (Figure 5A, CR). In a long-term growth experiment similar proliferation rates were observed with CR- and AS-GBP-1-transduced cells (seven cell passages at a 1:4 splitting ratio after 3 months of culture in the presence of puromycin), whereas S-GBP-1-transduced cells grew significantly less (three cell passages for the same period of time), indicating that GBP-1 inhibits cell growth. Figure 5.GBP-1 mediates the inhibition of endothelial cell proliferation in response to IC. (A) Schematic presentation of the retroviral expression vector pBabePuro (control, CR) containing the full-length GBP-1 cDNA in sense (S-GBP-1, S) or antisense (AS-GBP-1, AS) orientation. (B) Western blot analysis of GBP-1 expression. Polyclonal rabbit anti-GBP-1 peptide antibody (αGBP-1 pAb), polyclonal rabbit anti-GBP-1 antibody (αGBP-1 rAb), actin control (Actin). After transduction, HUVEC were grown for 10 days in 0.3 μg/ml puromycin and then incubated for 24 h in the presence or absence of IL-1β (20 U/ml). (C) Proliferation experiments with S-GBP-1- (white columns) and CR- (gray columns) transduced HUVEC in the presence of increasing concentrations of AGF, and (D) with AS-GBP-1- (black columns) and CR- (gray columns) transduced HUVEC in the presence of AGF and increasing concentrations of IL-1β. (E) In vitro adhesion assay with S-GBP-1-, AS-GBP-1- and CR-transduced HUVEC stimulated with increasing concentrations of IL-1β and U937 monocytes. In (C), (D) and (E) the mean values obtained in three different transduction experiments are shown. (F) Western blot analyses of phospho-ERK-1/2 (upper panel) and GBP-1 (lower panel) expression in S-GBP-1- and CR-transduced HUVEC stimulated with either VEGF (10 ng/ml), bFGF (10 ng/ml) or AGF (VEGF and bFGF, 10 ng/ml each) for 15 min, respectively. (G) TUNEL analyses (upper panels) and the respective phase contrast images (lower panels) of untreated S-GBP-1-, AS-GBP-1- and CR-transduced HUVEC and as a positive control of apoptotic CR-transduced HUVEC incubated for 24 h in the presence of TNF-α (30 U/ml). Download figure Download PowerPoint For a detailed analysis of cell growth under different conditions of stimulation, a short-term selection procedure (10 days) was used (see Materials and methods). After this time, GBP-1 protein synthesis was increased in IL-1β-treated control cells (Figure 5B, CR), but not in IL-1β-treated AS-GBP-1-expressing cells (Figure 5B, AS), demonstrating that expression of the endogenous GBP-1 gene was not impaired by the transduction procedure and that induction of the cellular GBP-1 protein was efficie
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