Tumor Necrosis Factor-alpha Regulates Expression of Vascular Endothelial Growth Factor Receptor-2 and of Its Co-receptor Neuropilin-1 in Human Vascular Endothelial Cells*

Enrico GiraudoDagger , Luca Primo, Enrica Audero, Hans-Peter Gerber§, Pieter Koolwijk, Shay Soker**, Michael KlagsbrunDagger Dagger , Napoleone Ferrara§, and Federico Bussolinoparallel

From the Vascular Biology Laboratory, Department of Genetics, Biology and Biochemistry, Medical School, University of Torino, Torino, 10126 Italy, the § Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080, the  Department of Vascular and Connective Tissue Research Gaubius, Laboratory TNO-PG, 2333 CK Leiden, The Netherlands, ** Departments of Urology and Surgery, Children's Hospital, Harvard Medical School, and the Dagger Dagger  Departments of Surgery and Pathology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Tumor necrosis factor-alpha (TNF-alpha ) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha -mediated pathological settings.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

A well regulated angiogenesis is critical for embryonic growth, bone remodeling, menstrual cycle, corpus luteum formation, and tissue repair. The stable vascular bed occurring in these physiologic conditions results from a balance of signals that favor angiogenesis and those that promote vascular regression. In contrast, a deregulated angiogenesis is pivotal in tumor progression and inflammatory and viral diseases (1-3). A number of naturally occurring growth factors can directly induce angiogenesis by stimulating endothelial cell proliferation and migration or act indirectly by triggering endothelial cells themselves or accessory cells (monocyte/macrophage, mastocytes, T cells) to release direct angiogenic inducers (1-3).

Tumor necrosis factor-alpha (TNF-alpha )1 is a powerful activator of angiogenesis in vivo in several animal models when used at low doses (4-6) but is inhibitory at high doses (7). However, the ability of TNF-alpha to induce in vitro biological responses related to angiogenesis is weak. TNF-alpha stimulates in vitro chemotaxis of bovine adrenal capillary endothelial cells (4) but inhibits wound repair (8) and is devoid of mitogenic activity (5). Angiogenesis promoted by TNF-alpha seems necessarily to be due to indirect effects. TNF-alpha activates in endothelial cells the synthesis of B61 (9), basic fibroblast growth factor (FGF) (10), and platelet-activating factor (11), all known to be angiogenic (6, 12), and of tissue factor (13), which is a regulator of vessel formation (14). In endothelial cells, TNF-alpha promotes the synthesis of urokinase-type plasminogen activator (15), which is involved in the progression phase of angiogenesis characterized by a remodeling of extracellular matrix proteins by proteolytic enzymes (Ref. 16; for reviews, see Refs. 1-3).

TNF-alpha cooperates with basic FGF, vascular endothelial growth factor-A (VEGF-A), and interleukin-8 to induce capillary-like tubular structure of human microvascular endothelial cell growth in a three-dimensional gel of extracellular matrix proteins (17, 18). In these systems, the type of extracellular matrix seems to address the features of the angiogenic model. TNF-alpha does not induce angiogenesis in vitro when the cells are plated on three-dimensional fibrin matrix, but it is permissive for the activity of basic FGF and VEGF-A. TNF-alpha up-regulates the activity of urokinase-type plasminogen activator, which is required for the formation of capillary structure in addition to the angiogenic molecules (17). Otherwise, TNF-alpha induces in vitro angiogenesis of endothelium plated on collagen type I. This activity is mediated by the release of VEGF-A, basic FGF, and interleukin-8 (18). Furthermore, TNF-alpha induces mesenchymal or tumor cells to release angiogenic molecules, including VEGF-A (7, 19). Finally, it has been reported that TNF-alpha regulates the expression of integrins involved in adhesion of endothelial cells to extracellular matrix and in angiogenesis (8, 20).

The puzzling effects of TNF-alpha on endothelial cells and new vessel growth suggest the presence of more than one angiogenic signaling pathway and that this cytokine may have different activities on endothelial cells depending on the microenvironment. In light of the relevance of the cooperation between TNF-alpha and VEGF-A (7, 17, 18) in angiogenesis, we studied the effect of TNF-alpha on the expression and function of VEGF receptors. Adult endothelial cells express on their surface VEGF receptor (VEGFR)-1 encoded by Flt-1 (21) and VEGFR-2 by KDR/Flk-1 (22, 23), but recent findings suggest that the latter alone is able to mediate the mitogenic and chemotactic effect of VEGF-A in endothelial cells (22, 24, 25). More recently, it has been reported that neuropilin-1, a receptor that mediates neuronal cell guidance (26), is expressed by endothelial cells and enhances the binding of VEGF-A165 isoform to VEGFR-2 (27). Here, we demonstrate that the pretreatment of endothelial cells with TNF-alpha is followed by an increased migration and wound repair induced by VEGF-A165. An augmented expression of VEGFR-2 and neuropilin-1 genes causes this effect.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Cultures-- Human umbilical vein endothelial cells, prepared and characterized as described previously (28), were growth in medium 199 (Life Technologies, Inc.) supplemented with 20% fetal calf serum (FCS) (Irvine, Santa Ana, CA), endothelial cell growth supplement (100 µg/ml), porcine heparin (50 units/ml), 100 units/ml of penicillin, and 100 µg/ml of streptomycin (all from Sigma), in gelatin (Life Technologies, Inc.)-coated tissue culture plates (Falcon, Becton Dickinson, Plymouth, UK). They were used at early passages (I-III). Human fibrosarcoma 8378 cells, which respond to TNF-alpha (29), were maintained in Dulbecco's modified Eagle's medium containing 10% FCS. Porcine aortic endothelial cells transfected with human VEGFR-2 (24) were cultured in Ham's F-12 (Sigma) supplemented with 10% FCS. Human foreskin microvascular endothelial cells isolated as described previously (17), were cultured on fibronectin-coated dishes in medium 199 buffered with 20 mM Hepes containing 10% human serum, 10% newborn calf serum, endothelial cell growth supplement (150 µg/ml), and porcine heparin (5 units/ml).

Experimental Design-- To verify the effects of TNF-alpha on the expression of VEGF receptors and on the biological activities elicited by VEGF-A165, the following experimental conditions have been used: confluent endothelial cell growth at a CO2 level of 5% in atmospheric air was treated with TNF-alpha (1 × 107 units/mg of protein; Genentech, Inc., San Francisco, CA) in medium 199 supplemented with 20, 5, and 1% FCS or 1% bovine serum albumin (BSA) (lipopolysaccharide-free, Sigma), twice washed with medium 199, and then used to extract RNA. Alternatively, cells were stimulated with VEGF-A165 (a gift of Dr. H. A. Weich, GBF, Braunschweig, Germany) (30) in medium 199 containing 1% FCS in chemotaxis or 3% BSA in wound healing experiments. In some experiments, endothelial cells were starved for 24 h in medium 199 containing 1% FCS and 1% BSA before adding TNF-alpha . The effect of mithramycin (Sigma), which inhibits gene expression by blocking Sp1 binding to the CG box (7, 31), was studied by treating the cells for 12 h in medium 199 containing 5% FCS with or without TNF-alpha .

RNA Extraction and Northern Analysis-- Total cellular RNA was isolated by guanidinium isothiocyanate extraction and centrifugation through cesium chloride (32). Equal amounts of total RNA (15 µg/lane) were electrophoresed in 1% agarose gels containing 6.3% formaldehyde in MOPS buffer (Sigma) and blotted on a Nylon Duralon-UV membrane (Stratagene) by the traditional capillary system in 10× SSC (1.5 M NaCl, 150 mM sodium citrate, pH 7) (32). Filters were cross-linked with UV light (0.5 J/cm2) and prehybridized for 4 h at 42 °C in 50% formamide deioinizate, 10% dextran sulfate, 1% SDS, 1 M NaCl, and 100 µg/ml denatured salmon sperm DNA. Hybridization was carried out overnight at 42 °C with [alpha -32P]dCTP-labeled (3000 Ci/mmol, Amersham, Buckinghamshire, United Kingdom, UK) human VEGFR-2 (a 0.729-kb HindIII-EcoRI of KDR cDNA) (23), VEGFR-1 (a 1.347-kb HindIII-BglII fragment of human FLT-1 cDNA) (21), neuropilin-1 (a 0.735-kb PstI-PstI fragment of human neuropilin-1 cDNA) (27) and beta -actin cDNAs (33). cDNAs were labeled using Rediprime random primer labeling kit (Amersham) according to manufacturer's instructions. Posthybridization washes were performed at high stringency (once in 2× SSC, 0.1% SDS for 30 min, once in 0.2× SSC, 0.1% SDS for 30 min and twice in 0.1× SSC, 0.1% SDS for 30 min) at 57 °C, and the membranes were exposed on autoradiography with Hyperfilm-MP (Amersham).

Nuclear Run-on-- Nuclei were isolated from cultured endothelial cells essentially according to Ref. 34. Briefly, cells (2 × 107 cells/assay) were washed twice with ice-cold phosphate-buffered saline (PBS), scraped and collected in a 15-ml centrifuge tube by centrifugation at 500 × g for 5 min at 4 °C. Subsequent steps were performed at 4 °C. The cells were resuspended in 4 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40) and allowed to stand on ice for 5 min. and then centrifuged at 500 × g at 4 °C for 5 min. Nuclei were resuspended in 200 µl of glycerol storage buffer (10 mM Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA) and frozen in liquid N2. In vitro transcription and isolation of the resulting nuclear RNA were performed as described by Ikeda et al. (35). Two-hundred µl of frozen nuclei were thawed and mixed with 200 µl of 2× reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl 10 mM dithiothreitol, 400 units/ml placental ribonuclease inhibitor (Stratagene), 20 mM creatine phosphate (Sigma), 200 µg/ml creatine phosphokinase (Sigma), a 1 mM concentration each of ATP, CTP, and GTP (Stratagene), and 100 µCi of [alpha -32P]UTP (3000 Ci/mmol, Amersham). Samples were incubated at 30 °C for 30 min with shaking and for 5 min in the presence of 20 units of DNase I (RNase-free, Life Technologies, Inc.). After the addition of proteinase K (150 µg/ml, Sigma) and SDS (0.5% final concentration), incubation was continued at 37 °C for 30 min. Extracted RNA was resuspended in TES buffer (10 mM TES, pH 7.4, 10 mM EDTA, 0.2% SDS) at 5 × 106 cpm/ml. Linearized plasmids containing the target cDNAs (15 µg) were immobilized onto a nylon Duralon-UV membrane (Stratagene) using a Bio-Dot SF microfiltration apparatus (Bio-Rad). The filters were prehybridized overnight at 42 °C with hybridization buffer containing 20 mM PIPES (Sigma), pH 6.4, 50% formamide (Sigma), 2 mM EDTA, 0.8 M NaCl, 0.2% SDS, 1× Denhardt's solution (0.02% Ficoll, 0.02% BSA, 0.02% polyvinylpyrrolidone), 200 µg/ml E. coli tRNA (RNase-free, Stratagene). Hybridization was at 42 °C for 48 h in the same solution supplemented with 15 × 106 total cpm of labeled RNA. The filters were washed twice in 2× SSC, 0.5% SDS at 42 °C for 30 min, twice in 0.3× SSC, 0.5% SDS, at 42 °C for 30 min and then incubated with 10 µg/ml RNase A in 2× SSC at 37 °C for 30 min. Further washed were done in 2× SSC at 37 °C for 30 min and then in 0.3× SSC at 37 °C for 30 min. The filters were exposed on autoradiography with Hyperfilm-MP and intensifying screens at -80 °C. The amount of VEGFR-2 mRNA was standardized by comparison with the amount of beta -actin mRNA. Densitometric analysis was performed with a GS250 Molecular Imager (Bio-Rad).

Western Blot-- Endothelial cells were washed twice with PBS and lysed on ice with 1 ml of 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 5 mM EDTA, 50 µg/ml pepstatin, 100 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 500 µg/ml soybean trypsin inhibitor (all from Sigma). After centrifugation (20 min at 4 °C at 13,000 × g), protein were solubilized, separated by SDS-polyacrylamide gel electrophoresis (7%), transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA), probed with rabbit anti-VEGFR-2 antibody (C-1158, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and detected by ECL (Amersham).

Binding Assay and Analysis-- Recombinant VEGF-A165 (2 µg) was dissolved in 200 µl of sodium phosphate buffer 20 mM, pH 7.4, and transferred in IODO-GEN-coated tubes (50 µg/ml) (Pierce), where VEGF-A165 was iodinated (5 min, 4 °C) with 1 mCi of 125I (Amersham). Twenty µl of phosphate buffer 20 mM, pH 7.2, containing 1% BSA, 0.4 M NaCl, 0.1% CHAPS (Pierce) was added, and the reaction products were separated on Sephadex-G10. The specific activity of the tracer was 90,000 cpm/ng. 125I-VEGF-A165 retained its biological activity as measured by migration of endothelial cells (28). For specific binding studies confluent cells plated in 24-well plates were incubated an orbital shaker at 4 °C for 2 h in 200 µl/well of binding medium (medium 199 containing 20 mM Hepes buffer, pH 7.4, 0.1% BSA, 100 µg/ml soybean trypsin inhibitor) with increasing concentrations of 125I-VEGF-A165 in the presence of a 100-fold excess of unlabeled ligand. Endothelial cells were washed three times with ice-cold PBS containing 0.1% BSA and lysed in 200 µl/well of SDS 2% in PBS. Lysates were counted using a Beckman gamma  5500B counter. Triplicate samples under each condition were obtained for each experiment. Specific binding, calculated subtracting from the total cpm bound after incubation with a 100-fold excess of unlabeled ligand, was approximately 80%. The Kd was estimated by Scatchard plot using the Ligand program (Elseviere-Biosoft, Cambridge, UK).

Transient Transfection of pGL2basicFLK Plasmid-- The 2.0-kb XhoI/SacI fragment of Flk-1 promoter (36) was subcloned in pGL2basic plasmid (Promega, Madison, WI) to generate the luciferase reporter vector pGL2basicFlk-1.2 Human fibrosarcoma 8387 cell line (2 × 105 cells/well) was transfected with 3 µg of pGL2basicFlk-1 or pGL2basic using Superfect Transfection Reagent according to the manufacturer's instructions (Qiagen, Inc., Valencia, CA).The generated plasmid-liposome complex in 0.6 ml of Dulbecco's modified Eagle medium containing 10% FCS was incubated with the cells for 3 h at 37 °C in 5% CO2. The medium was then replaced with fresh medium, and the cells were stimulated for 4 h with TNF-alpha (20 ng/ml) or left untreated. pSVgal construct (2.5 µg) was co-transfected to correct for the variability in transfection efficiency, and beta -galactosidase activity was assayed with chlorophenol red beta -D-galactopyranoside (Boehringer Mannheim GmbH, Mannheim, Germany) as a substrate. For final luciferase assay, cells were lysed in 0.2 ml of passive lysis buffer (dual luciferase assay, Promega) at 4 °C, and 20 µl of cleared (12,000 × g for 2 min at 4 °C) cell extract containing 50 µg of protein were mixed with 0.1 ml of luciferase assay buffer. Light production was measured for 5 s in a luminometer (Magic Lite Analyzer, Ciba Corning, Milano, Italy), and results were normalized to the beta -galactosidase activity.

Migration Assay-- Migration assay was performed as described previously with Boyden's chamber technique (28). Polycarbonate filters (5-µm pore size polyvinylpyrrolidone-free; Neuroprobe, Pleasanton, CA) were coated with 0.1% gelatin for 6 h at room temperature. VEGF-A165 in medium 199 supplemented with 1% FCS was seeded in the lower compartment of the chamber, and 1.25 × 105 resuspended cells in 50 µl of medium 199 containing 1% FCS were then seeded in the upper compartment. At the end of the incubation (37 °C in air with 5% CO2 for 6 h), filters were removed and stained with Diff-Quik (Baxter Spa, Rome, Italy), and 10 high power oil immersion fields were counted. The results obtained were analyzed by one-way analysis of variance and the Student-Newman-Keuls test (Statistic Software; Bio-Soft).

Wound Healing-- Human endothelial cells were grown at confluence on 24 wells and monolayers were treated for 24 h with TNF-alpha (10 ng/ml) or vehicle alone in medium 199 containing 5% FCS. After washes, the monolayer was wounded with a razor blade (lesion surface: 20 mm2) as described (28) and incubated in medium 199 containing 3% BSA with or without VEGF-A165. After 24 h, the cells were fixed and stained as described (28). To quantify the repair process, phase-contrast microscopic pictures of wounded monolayer were recorded with a still video camera recorder (R5000H; Fuji Photo Film Co., Tokyo, Japan), and cell number was counted in 10 fields of 1 mm2 randomly selected, with a Cosmozone image analyzer (Nikon, Tokyo, Japan).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Increase of VEGFR-2 and Neuropilin-1 mRNA in Endothelial Cells Challenged with TNF-alpha -- Several studies have shown that 7.0-kb VEGFR-2 (23, 37) and 7.5-kb VEGFR-1 mRNAs (21) are expressed constitutively by endothelial cells in culture (37, 38). The second band of 3.4 kb recognized by VEGFR-2 cDNA represents an alternative transcript, as previously reported (37). Furthermore, neuropilin-1 has been recently demonstrated to be a specific co-receptor of VEGFR-2 for the binding of VEGF-A165 isoform (27). TNF-alpha stimulation of endothelial cells from human umbilical cord for 24 h induced an increase in VEGFR-2 (Fig. 1) and neuropilin-1 (Fig. 3B) mRNA levels. The effect on VEGFR-2 transcript was observed in different conditions of culture: medium supplemented with 5% FCS (Fig. 1B), 20% FCS (Fig. 1C), or 1% FCS associated with 1% BSA (Fig. 1A). In medium 199 containing 20% FCS, TNF-alpha was active at 1 ng/ml. As shown in Fig. 2, the levels of VEGFR-2 mRNA began to increase after 4 h of incubation with TNF-alpha (20 ng/ml), reached a maximum level after around 24 h and was gradually reduced after 48 h. TNF-alpha was also active on foreskin microvascular endothelial cells. In the basal condition, these cells did not express amount of mRNA detectable by Northern technique. However, after treatment with TNF-alpha , the VEGFR-2 mRNA was evident in microvascular endothelial cells (Fig. 3A). The expression of VEGFR-1, the second receptor expressed on endothelial cell membrane, was not modified by TNF-alpha treatment (Fig. 3B).


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Fig. 1.   Effect of TNF-alpha on VEGFR-2 mRNA expression in human endothelial cells. Northern blot analysis of total RNA extracted from confluent endothelial cells stimulated for 24 h with TNF-alpha in medium 199 containing 1% FCS and 1% BSA (A), 5% FCS (B), or 20% FCS (C). Fifteen µg of total RNA were run in a formaldehyde-agarose gel and, after blotting to Duralon membrane, hybridized to VEGFR-2 cDNA labeled with [alpha -32P]dCTP. Transcripts have been visualized by autoradiography. The lower panel displays an image of the respective ethidium bromide-stained nylon membranes to demonstrate even loading and transfer. This experiment is representative of three performed with similar results.


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Fig. 2.   Time course of TNF-alpha -induced up-regulation of VEGFR-2 mRNA expression in human endothelial cells. A, VEGFR-2 mRNA level from starved and confluent endothelial cells stimulated with 20 ng/ml TNF-alpha in medium 199 containing 20% FCS was determined by Northern blotting as detailed in the legend to Fig. 1. B, an image of the respective ethidium bromide-stained nylon membranes to demonstrate even loading and transfer. This experiment is representative of two experiments performed with similar results.


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Fig. 3.   Effect of TNF-alpha on expression of VEGFR-2 mRNA in human foreskin endothelial cells (A), of neuropilin-1 mRNA (B), and of VEGFR-1 mRNA in human endothelial cells from umbilical cord (C). A, confluent human endothelial cells from vein cord (lanes 1 and 2) and from foreskin endothelium (lanes 3 and 4) were stimulated for 24 h with 20 ng/ml TNF-alpha in medium 199 containing 10% (foreskin endothelium) or 20% FCS (umbilical endothelial cells) (lanes 2 and 4) or vehicle alone (lanes 1 and 3). VEGFR-2 mRNA was determined by Northern blotting as detailed in the legend to Fig. 1. B, confluent human endothelial cells from vein cord were stimulated for 24 h with 20 ng/ml TNF-alpha in medium 199 containing 5% FCS (lane 2) or vehicle alone (lane 1). Neuropilin-1 mRNA was determined by Northern blotting as detailed in the legend to Fig. 1. C, confluent human endothelial cells from vein cord were stimulated for 24 h with 20 ng/ml TNF-alpha in medium 199 containing 5% FCS (lane 2) or vehicle alone (lane 1). VEGFR-1 mRNA was determined by Northern blotting as detailed in the legend to Fig. 1. The lower panels display an image of the respective ethidium bromide-stained nylon membrane to demonstrate even loading and transfer. These experiments are representative of two performed with similar results.

Since it has been reported that TNF-alpha reduces the VEGFR-2 mRNA expression in human endothelial cells (39), two Northern blots have been performed in two different laboratories (Dr. M. Introna, "Mario Negri" Institute, Milano, Italy; Dr. V. van Hinsbergh, TNO Prevention and Health, Leiden, The Netherlands) with results similar to those shown in Fig. 1. Furthermore, a marked increase of VEGFR-2 mRNA was also observed in porcine aortic endothelial cells transfected with the VEGFR-2 gene and stimulated with TNF-alpha (20 ng/ml) for 12 h (data not shown).

Induction of VEGFR-2 Gene Expression by TNF-alpha -- In order to investigate whether TNF-alpha activates transcription of the VEGFR-2 gene in endothelial cells, nuclear run-on assay was performed. Nuclei were prepared from endothelial cells cultured with medium alone and with 20 ng/ml of TNF-alpha for 4, 24, and 48 h, and RNAs transcribed from these nuclei were hybridized with VEGFR-2 cDNA. We observed that TNF-alpha increased the transcription rate of VEGFR-2, without affecting the transcription of beta -actin gene and of pBluescript plasmid, used as negative control (Fig. 4). The densitometric analysis done on three independent run-on assays showed that the transcriptional rate of the VEGFR-2 gene was respectively elevated of 1.2 ± 0.6-, 6.0 ± 0.5-, and 3.5 ± 0.3-fold after 4, 24, and 48 h of TNF-alpha incubation.


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Fig. 4.   Nuclear run-on analysis after exposure of endothelial cells to TNF-alpha . Nuclei were prepared from cells incubated in medium 199 containing 5% FCS with or without 20 ng/ml TNF-alpha . Transcription in the isolated nuclei was analyzed by hybridization of 32P-labeled RNA to 15 µg of VEGFR-2, beta -actin, and pBluescript cDNAs immobilized on nitrocellulose membrane. This experiment is representative of two experiments performed with similar results.

To further confirm that TNF-alpha regulates the transcription of VEGFR-2 gene, the mouse VEGFR-2 promoter-based reporter gene construct (36) was transiently transfected in 8378 human fibrosarcoma cells, which were treated for 4 h with TNF-alpha (20 ng/ml). This cell line was selected instead of endothelial cells, because it was more easily transfected with the construct than endothelial cells and expresses a functional active TNF receptor (29). Analysis of the respective luciferase expressions in untreated and TNF-alpha -stimulated cells revealed notable basal activity of the pGL2basicFlk-1 construct, which was increased after treatment with the cytokine by a factor of 2 (Fig. 5).


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Fig. 5.   Activity of VEGFR-2 promoter-based luciferase construct in human fibrosarcoma 8387 cell line. Cells were transfected with 3 µg of pGL2basicFlk or pGL2basic and subsequently treated for 4 h with 20 ng/ml TNF-alpha or vehicle alone. Extracts were analyzed for luciferase level. Transfection efficiency was corrected by cotransfection with pSVbeta gal. Mean ± S.D. of three experiments performed in duplicate is shown.

Human and mouse VEGFR-2 promoter contain five Sp1 elements (40), a transcription factor involved in TNF-alpha -induced gene expression in endothelial cells (41). We evaluated the role of Sp1 elements in the regulation of VEGFR-2 transcription by incubating endothelial cells with mithramycin, an inhibitor of the binding of the transcription factor to the CG box (7, 31). Fig. 6 shows that mithramycin at 1 and 10 nM inhibited the TNF-alpha -induced expression of VEGFR-2, suggesting that Sp1 binding sites in the promoter are involved in up-regulation of VEGFR-2 molecules.


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Fig. 6.   Effect of mithramycin on endothelial cell increase in VEGFR-2 mRNA levels by TNF-alpha . Cells were stimulated in medium 199 supplemented with 5% FCS with 20 ng/ml TNF-alpha in the absence or presence of 1 and 10 nM mithramycin. The cellular levels of VEGFR-2 transcript were determined by Northern blotting as detailed in the legend to Fig. 1. The lower panel displays the Northern blot performed with beta -actin cDNA. This experiment is representative of two experiments done with similar results.

TNF-alpha Increased the Expression of VEGFR-2 on Endothelial Cell Surface-- The effect of TNF-alpha on the up-regulation of VEGFR-2 expression was further investigated with the analysis of 125I-VEGF-A165-specific binding at equilibrium on the endothelial cell surface. Since TNF-alpha does not up-regulate VEGFR-1 (Fig. 3C), the binding studies were performed by incubating endothelial cells with 125I-VEGF-A165 concentrated to 50 pM or higher. This experimental condition excluded the analysis of the binding of VEGF-A165 to VEGFR-1 (42), which has a Kd ranging from 9 to 16 pM (24, 43), and indicated a single high affinity VEGF-A165 binding site on endothelial cells (Fig. 7A). Cell treatment for 24 h with TNF-alpha (20 ng/ml) in medium 199 containing 5% FCS produced a significant increase in 125I-VEGF binding to cell surface. Nonlinear regression analysis (Fig. 7B) of the data reported in Fig. 7A indicated a Kd = 137 ± 23 pM (n = 3) in untreated cells and a Kd = 122 ± 21 pM (n = 3) in TNF-alpha -treated cells. In contrast, TNF-alpha triplicates the number of binding sites expressed on cell membrane (in untreated cells, Bmax = 79 ± 14 fmol; in TNF-alpha -treated cells, Bmax = 243 ± 16 fmol, n = 3). Similar results have been obtained with TNF-alpha -treated endothelial cells in presence of 20% FCS (data not shown).


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Fig. 7.   Effect of TNF-alpha on binding of radiolabeled VEGF-A165 to human endothelial cells. A, specific ligand binding curve. Monolayers were incubated with TNF-alpha (20 ng/ml) (black-triangle) or vehicle alone (bullet ) for 24 h in medium 199 containing 5% FCS. After washes, cells were incubated with indicated concentrations of 125I-VEGF-A165 for 2 h at 4 °C in the presence of a 100-fold excess of cold ligand. B, Scatchard plot of the data reported in A. The data shown are representative of three experiments.

To analyze the expression of the protein encoded by VEGFR-2 gene, endothelial cells were treated with TNF-alpha for 24 h, and then the proteins from cell lysate were separated by SDS-polyacrylamide gel electrophoresis and probed with anti-VEGFR-2 antibodies. Fig. 8 shows that TNF-alpha treatment increased the amount of a 210-kDa protein recognized by an antibody anti-VEGFR-2. TNF-alpha did not modify the expression of proteins recognized by antibody anti-VEGFR-1 (data not shown).


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Fig. 8.   Western blot analysis of VEGFR-2 in human endothelial cells stimulated with TNF-alpha . Confluent endothelial cell monolayers were treated for 24 h with TNF-alpha in medium 199 containing 5% FCS. 200 µg of lysed proteins were separated by SDS-polyacrylamide gel electrophoresis (7%), blotted onto polyvinylidene difluoride membrane, probed with rabbit anti-VEGFR-2 antibody, and detected by enhanced chemiluminescence.

Effect of TNF-alpha on VEGF-A165-induced Endothelial Cell Migration-- VEGF-A165 induced in a dose-dependent manner the migration of endothelial cells as evaluated by the Boyden chamber technique in agreement with previous observations (24, 44). The maximal migration was obtained with a concentration of VEGF-A165 of 10 ng/ml (3.7-fold the control value, p < 0.05). TNF-alpha alone did not influence endothelial cell migration. However, when endothelial cells were stimulated for 24 h with TNF-alpha (20 ng/ml) in the presence of 20% FCS (Fig. 9) or lower FCS amounts (1 and 5%; not shown), they showed an increased motility after challenge with VEGF-A165. TNF-alpha treatment sensitized endothelial cells to an ineffective dose of VEGF-A165 (1 ng/ml) and was able to double the number of migrating cells stimulated with VEGF-A165 used at optimal concentration (10 ng/ml) (Fig. 9). The migration of endothelial cells triggered by hepatocyte growth factor (28) was not enhanced by cell treatment with TNF-alpha , suggesting that the effect of this cytokine is specific for VEGF-A165 (Table I).


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Fig. 9.   Effect of TNF-alpha on migration of human endothelial cells elicited by VEGF-A165. The migration of cells was measured by the modified Boyden chamber technique, as described under "Experimental Procedures." Human endothelial cells were treated with 20 ng/ml TNF or vehicle alone for 24 h in medium 199 with 5% FCS. Suspended cells (1.25 × 105) in medium 199 containing 1% FCS were seeded in the upper compartment of the chamber, and VEGF-A165 suspended in the same medium containing 1% FCS was placed in the lower compartment. Cells that migrated after 6 h of incubation to the lower surface of the filter were counted after coding samples. The numbers are the mean ± S.D. of eight experiments performed in triplicate. *, p > 0.05.

                              
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Table I
Effect of TNF-alpha on endothelial cell migration stimulated by VEGF-A165, and hepatocyte growth factor
Endothelial cells were pretreated with TNF-alpha (20 ng/ml) or vehicle alone for 24 h and then stimulated in Boyden's chamber with 10 ng/ml each molecule in medium 199 containing 1% FCS and processed as detailed in the legend of Fig. 1. The values are the means ± S.D. of three experiments done in triplicate.

Effect of TNF-alpha on VEGF-A165-induced Wound Repair in Endothelial Cell Monolayers-- A wound healing assay in vitro, i.e. the ability of filling artificial gaps created in cell monolayers, requires both cell growth and activation of cell movements. In preliminary experiments, we demonstrated that VEGF-A165 used at 10 ng/ml induced repair of mechanical wounds generated in human endothelial cell monolayers within 24 h but was ineffective at 1 ng/ml. The experiment given in Fig. 10 shows that pretreatment of the cells with TNF-alpha (20 ng/ml) for 24 h evidently enhances the VEGF-A165-induced wound repair. Table II provides a quantitative analysis of cells migration into and across the wound, supporting the qualitative experiment in Fig. 10.


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Fig. 10.   Effect of TNF-alpha on wound healing of human endothelial cell monolayer induced by VEGF-A165. Confluent endothelial cell monolayers were treated for 24 h with TNF-alpha (20 ng/ml) in medium 199 containing 5% FCS and washed twice with medium without FCS. The monolayer was wounded with a cross-shaped scratch and stimulated with VEGF-A165 (1 ng/ml) for 24 h in medium 199 containing 3% BSA. At the end of incubation, cells were fixed and stained by crystal violet. Magnification was ×2. The picture is representative of five experiments with similar results.

                              
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Table II
Effect of TNF-alpha on endothelial cell wound repair stimulated by VEGF-A165
Endothelial cell monolayer was treated with TNF-alpha (10 ng/ml) or vehicle alone for 24 h and then wounded with a razor blade. After washes, cells were stimulated with VEGF-A165 (1 ng/ml) in medium 199 containing 3% BSA and processed as described in the legend of Fig. 2. At the end of stimulation, the wounded area was recorded with a still video camera recorder. Cells in 10 fields of 1 mm2 were counted with an image analyzer. The values are the means ± S.D. of 10 fields in one experiment representative of three performed with similar results.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

TNF-alpha is an inflammatory cytokine with a wide spectrum of biological activities including angiogenesis (1, 45, 46). TNF-alpha acts particularly on the formation of new vessels by multiple indirect ways instead of promoting directly the sprout of endothelial cells and their growth, as the direct angiogenic inducers. The release of direct angiogenic molecules and up-regulation of proteolytic systems seem to be the biological events triggered by TNF-alpha to participate in angiogenesis (6, 7, 10, 12, 13, 17-19). In this report, we add a new piece to this mosaic, showing that TNF-alpha increases the transcription rate of the VEGFR-2 gene in vascular endothelial cells, resulting in augmented number of molecules expressed on cell surface and enhances the biological response of endothelial cells to VEGF-A165. This statement is based on five major observations: 1) in the binding analysis performed with 125I-VEGF-A165 concentrated to 50 pM or higher in order to render negligible the contribution of VEGFR-1 (42), TNF-alpha triples the number of high affinity binding sites for VEGF-A165 on endothelial membrane without affecting the affinity of the receptor for the ligand; 2) this effect is coupled to an early increase of mRNA expression of VEGFR-2, whereas the VEGFR-1 transcript is unchanged; 3) the up-regulation of VEGFR-2 mRNA results from an increase of the transcription as demonstrated by nuclear run-on assay and by the mouse VEGFR-2 promoter activation transfected in a human fibrosarcoma cell line responsive to and challenged with TNF-alpha ; 4) human endothelial cells pretreated with TNF-alpha are more responsive to VEGF-A165 than untreated cells in terms of migration and ability to repair a wounded monolayer; 5) the enhancement effect of TNF-alpha on endothelial cell migration is not observed when hepatocyte growth factor, an activator of endothelial motility (28), is used as stimulus, suggesting a relative specificity of the system.

The effect of TNF-alpha on mRNA expression of VEGFR-2 is dose-dependent and consistently detected with 1 ng/ml cytokine. The mRNA expression appears within 4 h after TNF-alpha stimulation and persists up to 24 h and then declines to basal level within 48 h. This time course is similar to that of other genes activated by TNF-alpha to direct endothelial cells toward a proinflammatory phenotype (47). Among these genes, E-selectin and vascular cell adhesion molecule-1 regulate leukocyte transmigration, but the soluble forms of the encoded proteins have been reported to be angiogenic too (48).

The control of VEGFR-2 transcription is entrusted by a promoter characterized by putative binding sites for AP-2, Sp1, and NF-kappa B transcription factors (36, 40). The TNF-alpha -induced activation of VEGFR-2 was inhibited by mithramycin, an inhibitor of Sp1 interaction with its consensus sequence (7, 31), suggesting that the rapid increase of VEGFR-2 might thus be mainly mediated through the activation of Sp1 in endothelial cells. Recently, it has been reported in endothelial cells that Sp1 is the major nuclear protein binding to VEGFR-2 promoter (49) and that TNF-alpha up-regulates Sp1 transcription and expression with a time course similar to that described in this study for VEGFR-2 (41). Sp1 oligonucleotide antisense inhibits the stimulating effect of TNF-alpha on VEGF-A production by endothelium and on in vitro angiogenesis (18). Since the VEGF-A promoter has Sp1 binding sites (50), Yoshida and co-workers have hypothesized that inhibition of VEGF-A synthesis may be the mechanism by which TNF-alpha -induced in vitro angiogenesis is affected (18). Viewed in light of the results shown here, it is also reasonable to explain the inhibition of Sp1 oligonucleotide antisense on angiogenesis as an effect on the mechanisms leading to the up-regulation of VEGFR-2 induced by TNF-alpha .

TNF-alpha also increases the transcription of neuropilin-1, which enhances the binding of VEGF-A165 to VEGFR-2 and VEGF-A165-mediated chemotaxis (27). The binding of VEGF-A165 to neuropilin-1 is mediated through the amino acid sequence encoded by exon-7, absent in other VEGF-A isoforms (25), suggesting the high specificity of this co-receptor for VEGF-A165 (27). Further experiments could discriminate whether the effect of TNF-alpha is restricted to VEGF-A165 or also present in other isoforms.

The observed up-regulation of VEGFR-2 by TNF-alpha is in disagreement with the results published by Patterson and co-workers (39), who demonstrated that TNF-alpha down-regulates VEGFR-2 expression in human endothelial cells from veins or arteries. This discrepancy could be due to differences in experimental conditions. However, human microvascular endothelial cells from omental tissue increase VEGFR-2 mRNA when challenged with TNF-alpha (18).

Therefore, this study brings new insight into the conditions regulating the endothelial cell response to VEGF-A165. Previous studies on the mechanism responsible for the regulated expression of VEGFR-2 have focused on TGF-beta 1 (51) or hypoxia (42). Notably, neutralizing anti-TNF-alpha antibodies did not neutralize the up-regulation of VEGFR-2 by conditioned media from hypoxic cells (42). Hypoxia does not affect directly the VEGFR-2 promoter (36), but the up-regulation is mediated by an unknown factor present in ischemic tissues (42). TGF-beta 1 decreases the expression of VEGFR-2 after a prolonged time of incubation, by a presently unknown molecular mechanism (51). Indeed, TNF-alpha is the first identified cytokine that increases the endothelial cell response to VEGF-A165 by a direct effect on the VEGFR-2 transcription.

Our in vivo preliminary experiments agree with our in vitro results. The treatment of DBA2 mice with TNF-alpha (750 ng intraperitoneally followed by a second dose 3 days after) allows an ineffective angiogenic concentration of VEGF-A165 to promote vascularization in a Matrigel plug (52) injected subcutaneously on the day of the second TNF-alpha injection (vehicle-treated mice were injected with 5 ng of VEGF-A165/0.75 ml of Matrigel (n = 5), and vascularized area was 6 ± 4% of total Matrigel area; vehicle-treated mice were injected with 0.75 ml of Matrigel (n = 3), and vascularized area was 3 ± 2% of total Matrigel area; TNF-alpha -treated mice were injected with 5 ng of VEGF-A165/0.75 ml of Matrigel (n = 5), and vascularized area was 34 ± 10% of total Matrigel area; TNF-alpha -treated mice were injected with 0.75 ml of Matrigel (n = 5), and vascularized area was 7 ± 3% of total Matrigel area). Involvement of VEGFR-2/VEGF-A system has recently been demonstrated in vivo in cancer disease and in chronic inflammation (44, 53, 54), processes in which TNF-alpha is markedly up-regulated (55-57). Furthermore, we have recently demonstrated that VEGFR-2 is also the receptor of Tat (58), a protein of immunodeficiency virus-1 involved in the angiogenesis associated with Kaposi's sarcoma (59). Since our results demonstrate that TNF-alpha presents as a potent inducer of VEGFR-2 synthesis, the TNF-alpha -mediated up-regulation of the unique VEGF receptor capable to mediate mitogenic and motogenic signals inside endothelial cells (22, 24) is likely to play an important role in the initiation and maintenance of angiogenesis and increased vascular permeability in these conditions.

    ACKNOWLEDGEMENTS

We thank Dr. Lena Claesson-Welsh (Uppsala University, Uppsala, Sweden), Dr. Martino Introna (Istituto "Mario Negri," Milano, Italy), and Dr. P. M. Comoglio (Institute for Cancer Research, Torino, Italy), who provided porcine aortic endothelial cells transfected with VEGFR-2, human fibrosarcoma 8378 cells, and human recombinant hepatocyte growth factor, respectively. Dr. Martino Introna and Victor van Hinsbergh provided mRNA of TNF-alpha -stimulated and control endothelial cells. VEGFR-2 and VEGFR-1 were kindly provided by Dr. Bruce Terman (Wyeth Ayerst Research, Pearle River, NY), and recombinant VEGF-A165 was provided by Dr. Herbert Weich (GBF, Braunschweig, Germany).

    FOOTNOTES

* This study was supported by grants from the European Community (Biomed-2 Project: BMHL-CT96-0669), the Italian Association for Cancer Research, Istituto Superiore Ri Sanità (X AIDS Project, Multiple-Sclerosis Project, Program on Tumor Therapy), the Dutch Cancer Society (TNOP 97-1511), National Institutes of Health (CA 37392 and 45548), Centro Nazionale Ricerche (P. F. Biotecnologie), and Ministero Dell' Universita e Della Ricerca Scientifica Technologica (60%).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a grant from the Italian Foundation for Cancer Research.

parallel To whom correspondence should be addressed: Dept. of Genetics, Biology and Biochemistry, Via Santena 5bis., 10126 Torino, Italy. Tel.: 39-11-6706684; Fax: 39-11-6635663; E-mail: Bussol{at}molinette.unito.it.

The abbreviations used are: TNF-alpha , tumor necrosis factor-alpha ; BSA, bovine serum albumin; FCS, fetal calf serum; FGF, fibroblast growth factor; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; kb, kilobase pair(s); MOPS, 3-(N-morpholino)propanesulfonic acidTES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidPIPES, piperazine-N,N'-bis(2-ethanesulfonic acid)CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

2 H. Gerber and J. Park, personal communication.

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