Departments of Exploratory Science, Molecular Genetics and Protein Engineering, Biogen Inc., 12 Cambridge Center, Cambridge, Massachusetts 02142, USA
*Author for correspondence (e-mail: Aniela_Jakubowski{at}Biogen.com)
Accepted September 24, 2001
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SUMMARY |
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Key words: TWEAK, Tumor necrosis factor-related, Angiogenesis, Basic fibroblast growth factor, Vascular endothelial growth factor
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Introduction |
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A number of angiogenic regulators belong to the tumor necrosis factor (TNF) family. Ligands of this family are expressed as type II membrane proteins, which may be proteolytically cleaved to produce soluble cytokines (Smith et al., 1994). These ligands trigger biological activities by binding and signaling through their corresponding receptors in the TNF receptor family. The majority of the TNF family members mediate host defense, inflammation and immunological regulation (Locksley et al., 2001
). In addition, some of these ligands regulate EC functions. For example, Fas/Fas ligand (FasL) interaction can induce endothelial capillary tube formation in vivo (Biancone et al., 1997
), while vascular endothelial growth inhibitor (VEGI) or TL1 inhibits EC survival and proliferation in vitro (Yue et al., 1999
; Zhai et al., 1999
). TNF-
also modulates EC behavior; however, its effects are complex. TNF-
inhibits EC growth yet induces capillary tube formation in vitro (Frater-Schroder et al., 1987
; Yoshida et al., 1997
). It also can be antiangiogenic in the context of solid tumors (Ruegg et al., 1998
) or angiogenic in corneal settings in vivo (Fajardo et al., 1992
; Frater-Schroder et al., 1987
; Leibovich et al., 1987
; Yoshida et al., 1997
).
TWEAK is a novel member of the TNF ligand family (Chicheportiche et al., 1997) that is broadly expressed at the mRNA level in many normal tissues and in nonlymphoid tumor cell lines. TWEAK was named for its TNF-relatedness, proinflammatory properties and weak ability to induce cell death (TNF weak). Although TWEAK protein expression and its physiological role(s) are still undefined, transfection studies suggest that TWEAK can function as a secreted cytokine, like TNF-
and Lymphotoxin
(LT
). The nature of the TWEAK receptor also remains controversial. One report described a biochemical association between TWEAK and death receptor-3 (DR-3) (Marsters et al., 1998
), but this interaction has not been confirmed (Kapstein et al., 2000
). In addition, TWEAK induces death in cells that lack DR3 mRNA (Schneider et al., 1999
), suggesting the existence of an unidentified TWEAK receptor distinct from DR3. Accumulating evidence supports the original characterization of TWEAK as a proinflammatory cytokine. Recently, TWEAK was reported to be expressed by
-interferon (IFN
)-stimulated human peripheral blood monocytes and to play a role in monocyte cytotoxicity against tumor cells (Nakayama et al., 2000
). TWEAK has also been shown to induce interleukin 8 (IL-8) in some tumor cell lines (Chicheportiche et al., 1997
) and IL-6 and IL-8 in human astrocytes in vitro (Saas et al., 2000
). However, additional data have emerged indicating the proangiogenic potential of TWEAK. TWEAK was reported to induce the proliferation of human ECs and smooth muscle cells and neovascularization when implanted in rat corneas (Lynch et al., 1999
), suggesting a positive role for TWEAK in vasculature formation in vivo.
In this study we further investigated the angiogenic potential of TWEAK. Our results demonstrate that TWEAK strongly promotes EC survival. Surprisingly, our results also show that TWEAK can be proangiogenic or anti-angiogenic depending upon the context. TWEAK promotes the proliferation, migration and morphogenesis of capillary lumens induced by basic fibroblast growth factor (bFGF). Conversely, TWEAK opposes EC morphogenic responses induced by vascular endothelial growth factor (VEGF). These results indicate that TWEAK may be an important angiogenic regulator that can modulate EC responses to other key angiogenic cytokines. Potential mechanisms underlying the bidirectional effects of TWEAK are discussed.
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Materials and Methods |
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Reagents and antibodies
Recombinant human bFGF was obtained as a supplier growth supplement (Clonetics), and bFGF and VEGF were purchased from R&D Systems (Minneapolis, MN) or Sigma (St Louis, MO). Annexin V-FITC and propidium iodide (PI) were from Pharmingen. Neutralizing mouse anti-human TNF and mouse anti-human IL-8 mAbs from R&D (Minneapolis, MN) and isotype-matched control Ig MOPC 21 (ICN Biomedicals Inc., Irvine, CA) were used for blocking studies. Biotin-conjugated anti-FLAG was from Eastman Kodak Company (New Haven, CT), and Phycoerythrin-Streptavidin (PE-Streptavidin) was from Southern Biotechnology Associates, Inc. (Birmingham, AL).
TWEAK-specific monoclonal antibodies
BE.B3, AB.G11 and AB.D3 were generated in Armenian hamsters using immunization with soluble human TWEAK protein and standard hybridoma generation procedures. The ability of AB.D3 and AB.G11 to bind to human and murine TWEAK, and the ability of BE.B3 to bind to human TWEAK, was demonstrated in an ELISA assay using recombinant soluble TWEAK proteins immobilized on 96-well microtiter plates. AB.D3 and AB.G11, but not BE.B3, were shown to have blocking activity on the basis of their ability to inhibit soluble FLAG-tagged human TWEAK binding to HT29 cells in a FACS analysis. BE.B3 was biotinylated using ImmunoPure Biotinylation kits following the manufacturers protocol (Pierce, Rockford, IL). A hamster control Ig (clone Ha4/8-3.1) was obtained from the American Type Culture Collection, and the mAb was purified from culture supernatant using a Protein A Fast Flow column (Pharmacia, Piscataway, NJ).
Recombinant soluble human TWEAK protein
Recombinant soluble human TWEAK (GenBank accession number AF030099), containing amino-acid residues A106-H249, was expressed either with or without an N-terminal FLAG epitope in the yeast Pichia pastoris (Carlsbad, CA). The fermentation medium from Pichia expressing soluble human TWEAK was concentrated and dia-filtrated in 20 mM Tris-HCl, pH 8.0 and ion-exchanged on a Q Sepharose column prior to loading onto a Zn chelating column. The soluble TWEAK was eluted using an imidazole gradient in 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.5 prior to final fractionation by size exclusion using a Sephacryl 300 column.
Analysis of apoptosis
1x105 HUVEC or HDMEC were seeded in six well plates and incubated overnight in CS-C complete medium. Following a wash in PBS, cells were cultured in CS-C complete media or incomplete media (supplemented with 0% FBS and 0.1% bovine serum albumin (BSA) or 2% FBS) with or without VEGF (10 ng/ml) or TWEAK (200 ng/ml). Where indicated, 2 µg/ml of anti-TWEAK mAb AB.G11 or control Ig was also added. After 48 hours, cells were washed with PBS and detached by incubation with dispase (CS-C) for 15 minutes at 37°C followed by replacement with PBS containing 5mM EDTA and 0.1% BSA for 15 minutes at 37°C. After an additional wash in PBS, cells were stained with FITC-Annexin-V and 5 µg/ml PI according to the supplier. Fluorescence was analyzed using FACStarPLUS (Becton Dickinson, San Jose, CA). Results are reported as the frequency of viable cells (Annexin and PI negative) and apoptotic cells (Annexin positive, PI negative and positive).
Proliferation assay
HUVEC in 96 well microtiter plates at subconfluence (4300 cells per well) were cultured overnight in CS-C medium. The medium was replaced with complete medium or with basal medium with or without BBE, TWEAK (100-200 ng/ml), a 1/500-1/1000 dilution of the bFGF supplement (Clonetics) or 1 ng/ml bFGF (R&D Systems), VEGF (10 ng/ml) or combinations of these factors. Where indicated, 10 µg/ml anti-TWEAK mAbs AB.D3, BE.B3 or hamster control Ig Ha4/8 were also added. Following a three day incubation, proliferation was measured by a 10 hour pulse with [3H]-thymidine.
Endothelial wound repair assay
A standard wound repair assay was employed as previously described (Morales et al., 1995). Briefly, a confluent monolayer of HUVEC was grown in CS-C complete medium in 35x10 mm cell culture dishes with 2 mm grids (Nalge Nunc International, Naperville, IL). The monolayer was wounded by two perpendicular strokes across the diameter of the dish with a 1 mm tip. The consistency of this methodology was shown by directly quantifying the width of the starting wound using a computerized measuring tool; variation was only 7%. Dislodged cells were aspirated, and plates were rinsed with PBS. Cells were cultured for 18 hours in complete media or in basal media as defined above with or without TWEAK (200 ng/ml), bFGF (1/1000 or 1 ng/ml), VEGF (10 ng/ml) or combinations of these before fixing with 1% paraformaldehyde followed by staining with Harris Hematoxylin (Sigma, St Louis, MO). Wound repair was quantified by visually counting the number of grids in which the gap was obscured by migrating cells. This number was divided by the total number of grids that lined the wound, and the results were expressed as mean percentage wound repair ± s.e.m.
Immunofluorescent staining
Cells were analyzed for TWEAK binding by incubation with FLAG-TWEAK, and binding was detected with either biotinylated mouse anti-FLAG mAb or biotinylated BE.B3 and streptavidin-PE. Cold competition was performed with FLAG-TWEAK at 100 ng/ml and increasing concentrations of nontagged TWEAK, and binding was detected with the biotinylated mouse anti-FLAG mAb. Blocking of TWEAK binding by the AB.D3 Ab was performed by preincubation of FLAG-TWEAK with 10 µg/ml of mAb.
Capillary tube formation assay
Capillary tube formation by ECs was analyzed using a fibrin matrix gel assay based on a method that had been previously described (Mach et al., 1999). Briefly, 4 mg/ml plasminogen-free human fibrinogen (Calbiochem, San Diego, CA) was dissolved in serum-free EBM-2 media with heparin and polymixin B both at 1 µg/ml (Sigma) as well as with the supplier supplements except for VEGF and bFGF. The fibrin solution was filter sterilized, and fibrin matrices were prepared by adding thrombin (20-50 milliunits/ml) (Sigma) and distributing 300 µl per well in 24 well plates. HUVEC (4x104 cells/cm2) were seeded onto the gel surfaces and overlayed with EBM-2 media as above, plus 5% FBS in the presence or absence of TWEAK, bFGF, VEGF or combinations of these factors. TWEAK was used at 1 µg/ml or 100 ng/ml, bFGF at 100 ng/ml and VEGF at 50 ng/ml. In some experiments, neutralizing mAbs specific for TNF (1 µg/ml) and IL-8 (10 µg/ml) or isotype control Ig were used. After 48-72 hours of culture, phase-contrast photomicrographs of the gel surface were taken. Gels were examined for the number of capillary sprouts by a blinded investigator. Gels were fixed with 10% ethanol for 10 minutes, transferred from the original wells to new wells, fixed with 4% paraformaldehyde, embedded in paraffin, cross sectioned (5 µm sections) and stained with hematoxylin and eosin.
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Results |
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Differential effect of TWEAK on EC morphogenesis induced by bFGF and VEGF
The growth of microvessels involves the coordinated proliferation, migration and morphogenetic organization of ECs into capillary tubes. The effect of TWEAK on morphogenic activity was assessed using EC cultures seeded onto the surface of fibrin gels in the presence or absence of bFGF or VEGF. We found that bFGF, but not TWEAK alone, induced morphological changes in the EC monolayer on the fibrin gel surface as evidenced by phase-contrast microscopy (Fig. 4A). Interestingly, addition of TWEAK to bFGF significantly enhanced these morphogenic changes, inducing about a two-fold increase in the number of capillary sprouts (Fig. 4A,C). Furthermore, histological analysis of cross-sections perpendicular to the matrix surface revealed that bFGF and bFGF + TWEAK, but not TWEAK alone, also promoted EC invasion into the fibrin matrix. However, only TWEAK + bFGF resulted in the formation of lumen-containing structures (Fig. 4B). Specifically, we counted 17 such structures in a total of 30 fields in cross-sections from the TWEAK + bFGF treated cultures versus one lumen-containing structure in those from cultures with bFGF alone. Similar results were obtained with several different EC types, including HUVECs, HDMEC, human pulmonary artery EC and human lung microvascular EC; the effect of TWEAK was specifically blocked by anti-TWEAK mAbs. No stimulation of lumen morphogenesis was observed when TWEAK was substituted for by another TNF family member, CD40L (data not shown). Since TWEAK has been shown to induce the production of IL-8 and TNF in cultured cell lines (Chicheportiche et al., 1997; Schneider et al., 1999
), we examined whether the cooperation between TWEAK and bFGF was a secondary effect involving these angiogenic modulators (Koolwijk et al., 1996
; Yoshida et al., 1997
). As shown in Fig. 4C, the number of capillary sprouts formed by TWEAK + bFGF was not reduced in the presence of neutralizing mAbs for these factors, indicating that neither TNF
or IL-8 are required for the cooperative effect. TNF was produced at <1 ng/ml and IL-8 at 1-2 ng/ml in these cultures, and the ability of the respective mAbs to neutralize these concentrations of TNF and IL-8 independently was confirmed by inhibition of TNF-induced tumor cell killing (data not shown) and neutralization of IL-8 in a cell culture model (Terui et al., 1998
).
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Discussion |
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Endothelial survival is critical for a sustained angiogenic process, which includes vessel sprouting, maturation and stabilization, and for long-term maintenance of newly formed vessels (Carmeliet, 2000). Factors such as VEGF, bFGF and Angiopoietin-1 have been shown to promote EC survival upon withdrawal of nutrients or serum (Nor et al., 1999
). Using similar conditions, we demonstrate that TWEAK also promotes survival of EC. In addition, we found that TWEAK markedly enhances EC proliferation in BBE-supplemented media, in complete agreement with previously reported results (Lynch et al., 1999
). In contrast, the effect of TWEAK alone, assessed here for the first time, was not significant. Importantly, using more defined culture conditions our studies reveal that TWEAK enhances EC proliferation induced by bFGF. Thus the proliferative activity of TWEAK under previous conditions presumably reflects its cooperativity with bFGF or other components. It is also possible that cooperativity between TWEAK and another cytokine/growth factor underlies the stimulatory effect of TWEAK observed in the corneal micropocket assay (Lynch et al., 1999
). which is characterized by post-surgical inflammation. TWEAK also enhanced bFGF-dependent EC wound repair. This effect is probably due to stimulation of EC migration rather than proliferation, as we found no increase in the number of cells recovered (relative to input) after one day of culture with bFGF alone or bFGF + TWEAK (data not shown). We also found no changes in EC-surface expression of a variety of integrins such as
v,
1,
2,
5, ß3 and ß1, which have been implicated in angiogenesis (Eliceiri and Cheresh, 1999
; Senger et al., 1997
), in cultures with the TWEAK/bFGF combination as compared with either agent alone (data not shown). The enhanced organization of ECs into lumen-containing structures within the fibrin gel matrix suggests an additional level of cooperation between TWEAK and bFGF, perhaps through pathways that mediate EC invasion, polarity or the formation of tight intercellular junctions. We propose two possible models to explain the cooperativity between TWEAK and bFGF. bFGF may be required to upregulate the TWEAK receptor, enabling TWEAK to induce a range of angiogenic activities. Alternatively, signaling through the TWEAK receptor may amplify or be amplified by bFGF-induced signals. Interestingly, although the identity of the TWEAK receptor is yet to be reported, our preliminary data indicate that it can activate NF
B and MAP kinase pathways in ECs (data not shown). Further experiments are required to define the mechanism(s) underlying the cooperativity between TWEAK and bFGF, and this will be the subject of future studies.
In contrast with the pro-angiogenic activity of TWEAK in the context of bFGF, we found that TWEAK can inhibit the angiogenic behavior of ECs in other settings. We observed TWEAK inhibition of EC morphogenesis induced by VEGF. We hypothesized that TWEAK might inhibit EC morphogenesis, but not survival or proliferation induced by VEGF, by reducing the expression of VEGF receptors, particularly Flt-1 (VEGFR-1), on the basis of the phenotype of Flt-1-deficient mouse embryos whose ECs fail to organize into tube-like structures (Korpelainen and Alitalo, 1998; Fong et al., 1995
). In addition, previous studies have indicated that Flt-1 mediates chemotactic but not proliferative responses, whereas the Flk-1/KDR receptor (also called VEGFR-2) mediates both proliferative and chemotactic signaling (Thomas, 1996
). However, we found only a modest 30% reduction in the expression of Flt-1 mRNA and no detectable reduction in mRNA for other angiogenic molecules, including Flt-4, TIE, TIE2, thrombin receptor, CD31 and endoglin, as measured in EC cultures with or without TWEAK, VEGF or VEGF + TWEAK (data not shown). It will be important to examine the effect of TWEAK on Flt-1 protein levels. We also analyzed the cell-surface expression of a variety of integrins (as above) and found them to be similar in cultures treated with TWEAK and VEGF as compared to VEGF alone (data now shown). Further analyses are required to delineate the mechanism of inhibition by TWEAK of VEGF-induced morphogenesis.
The dual role of TWEAK as an angiogenic regulator is reminiscent of the context-dependent role of TNF. Although the role of TWEAK as an angiogenic regulator cannot be deduced fully from in vitro studies, our data, together with the TNF paradigm, support the hypothesis that TWEAK can differentially modulate angiogenesis depending upon the particular angiogenic setting. TWEAK is ubiquitously expressed at the mRNA level. However, we have not detected TWEAK protein in normal tissues, tumor cell lines or HUVECs to date. The reported expression of TWEAK protein by IFN-stimulated human monocytes (Mach et al., 1999
) supports the possibility that angiogenesis may be modulated by TWEAK produced by infiltrating macrophages in the context of inflammatory responses.
In summary, our studies reveal a dual role for TWEAK in angiogenic regulation. We demonstrate that TWEAK promotes EC survival and regulates EC proliferation, migration, and morphogenesis in vitro through modulation of EC responses to bFGF and VEGF. These data suggest that TWEAK may play a role in the regulation of microvascular growth, remodeling, and/or maintenance in vivo, being pro-angiogenic or anti-angiogenic depending on the angiogenic microenvironment. Agonists or antagonists of the TWEAK pathway may provide useful therapeutic approaches to treatment in settings of ischemic injury, cancer, angioproliferative and inflammatory disorders.
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ACKNOWLEDGMENTS |
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