Vascular endothelial growth factor (VEGF) is a dimeric angiogenic factor that is overexpressed by many tumors and stimulates tumor angiogenesis. VEGF initiates signaling by dimerizing the receptors VEGFR-1 and VEGFR-2. The Fas receptor stimulates apoptosis, and artificial dimerization of the Fas cytoplasmic domain has been shown to induce apoptosis. We constructed a chimeric receptor (VEGFR2Fas) combining the extracellular and transmembrane domains of VEGFR-2 with the cytoplasmic domain of Fas receptor. When VEGFR2Fas was stably expressed in endothelial cells in vitro, treatment with VEGF rapidly induced cell death with features characteristic of Fas-mediated apoptosis. These findings demonstrate that VEGFR2Fas functions as a VEGF-triggered death receptor and raise the possibility that introduction of VEGFR2Fas into tumor endothelium or tumor cells in vivo may convert tumor-derived VEGF from an angiogenic factor into an antiangiogenesis agent.

VEGF,8 also known as VEGF-A or vascular permeability factor, is a secreted, dimeric angiogenic growth factor (1). VEGF is a key regulator of embryonic, physiological, and pathological angiogenesis and is believed to be a central mediator of tumor angiogenesis (1, 2). This is supported by the observation that VEGF expression is up-regulated in many human and experimental animal tumors (3, 4) and by the finding that interventions that block VEGF expression or activity inhibit tumor growth in animals (5, 6). In light of the importance of angiogenesis in tumor growth, and the central role of VEGF in tumor angiogenesis, many laboratories are investigating antiangiogenesis cancer therapies directed at VEGF or its receptors. These include techniques designed to reduce expression of VEGF or its receptors, to block binding of VEGF to its receptors, or to block signaling by VEGF receptors (5, 6). All of these approaches are designed to inhibit the angiogenic activity of VEGF secreted by tumor cells and infiltrating stromal cells. An alternative approach that has not been explored would be to use tumor-derived VEGF as a signal to directly induce apoptosis of tumor vessels or tumor cells themselves. This might be possible if VEGF receptor signaling could be reversed to generate apoptotic signals, rather than the endothelial survival and proliferation signals that are normally generated by endogenous VEGF receptors. The approach we have taken to create a VEGF-triggered death receptor is to modify a VEGFR such that on VEGF binding, it activates endogenous caspase-mediated apoptotic signaling pathways.

VEGF is secreted as a covalently linked homodimeric polypeptide (1). VEGF binds to two receptor tyrosine kinases, VEGFR-1 (also known as Flt1) and VEGFR-2 (Flk1/KDR), that are expressed primarily in endothelial cells, and some VEGF isoforms also bind to the nonkinase receptors neuropilin-1 and neuropilin-2 (7, 8, 9, 10). VEGF stimulates homodimerization and heterodimerization of VEGFR-1 and VEGFR-2, which become activated by autophosphorylation and activate downstream signaling pathways that mediate angiogenesis (7, 8, 11).

The Fas receptor (CD95/APO-1) is a cell surface death receptor that regulates lymphoid homeostasis (12). Fas receptor has an extracellular ligand-binding domain and an intracellular “death domain.” Fas-mediated apoptosis is initiated when Fas ligand stimulates trimerization or oligomerization of Fas receptor (12). The Fas-associated death domain adapter protein then binds to the cytoplasmic death domain of Fas and recruits and activates procaspase-8, an initiator caspase. Activated caspase-8 cleaves and activates downstream effector caspases that effect apoptosis either by cleaving intracellular proteins or by activating other proteases (12). Fas apoptotic signaling is inhibited by the binding of FAP-1 phosphatase to the COOH terminus of Fas (13). Endothelial cells express Fas, and Fas activation stimulates endothelial apoptosis (14). Fas ligand is trimeric, and it is generally thought that the Fas receptor must become trimerized or clustered to initiate apoptosis (12, 15). However, artificial dimerization of Fas cytoplasmic domains using chemical inducers of dimerization was found to activate Fas, suggesting that in some contexts, dimerization of the Fas cytoplasmic domain is sufficient to activate Fas (16). This finding suggested that a chimeric receptor composed of the extracellular VEGF-binding domain of VEGFR-2 and the cytoplasmic domain of Fas, which would be expected to dimerize on VEGF binding, might function as a VEGF-triggered apoptosis receptor.

Cell Culture and Growth Factors.

PAE cells were cultured in Ham’s F-12 medium supplemented with 10% calf serum and penicillin/streptomycin. PA317 amphotropic retroviral packaging cells were cultured in DME-H21 with 10% calf serum and pen/strep. Human VEGF-A165 was purchased from Peprotech. Human M-CSF, human PlGF-129, and human VEGF:PlGF (VEGF-A165:PlGF-129) were purchased from R&D Systems.

VEGFR2Fas and FmsFas Chimeric Receptor Constructs and Generation of Cell Lines.

VEGFR2Fas consists of the first 787 amino acids of mouse VEGFR-2, which includes the complete extracellular ligand-binding domain and entire transmembrane domain, fused to the complete 144 amino acid cytoplasmic domain of human Fas, beginning with Lys191. Human Fas cDNA was generously provided by Dr. S. Nagata. The Fas cytoplasmic domain cDNA was modified by PCR-directed mutagenesis, with an RsrII site added on the 5′ end to allow fusion at an RsrII site at the 3′ end of the VEGFR-2 transmembrane domain, as well as a hemagglutinin tag (SYDVPDYASLGGPS) on the COOH terminus. The amino acid sequence at the junction of VEGFR-2 and Fas is … VILVRTVKRKE … (VEGFR-2 sequence italicized). VEGFR2Fas(lprcg), which contains the lprcg point mutation in the Fas death domain, was made by PCR-directed point mutagenesis. To make FmsFas, an Rsr II site was added by PCR to the 3′ end of the human Fms transmembrane domain. The Fms extracellular and transmembrane domains were then fused to the Rsr II-Fas-HA fragment described above to create FmsFas. The amino acid sequence at the junction of Fms and Fas is … LLLLRTVKRKE … (Fms sequence and three amino acids encoded by RsrII site are italicized). The VEGFR2Fas receptor cDNA was subcloned into the replication-defective retroviral vector pMX1112, which confers hygromycin resistance, and the FmsFas cDNA and VEGFR2Fas(lprcg) cDNA were subcloned into the replication-defective retroviral vector LNCX, which confers G418 resistance. pMX/VEGFR2Fas, LNCX/FmsFas, and LNCX/VEGFR2Fas(lprcg) plasmids were separately transfected by calcium-phosphate precipitation into PA317 amphotropic retroviral packaging cells. To make control endothelial cells, PA317 cells were transfected with empty pMX1112 vector. Transfected PA317 cells were selected in hygromycin (400 μg/ml) or G418 (500 μg/ml) until colonies grew and then maintained as a pool. PA317 supernatant containing virus was filtered (0.4 μm) and added to PAE cells with Polybrene (8 μg/ml) for 6 h. PAE clones expressing VEGFR2Fas were obtained by selection in hygromycin (100 μg/ml) and cells expressing VEGFR2Fas(lprcg) by selection in G418 (250 μg/ml). Stably transfected clones were screened for receptor expression by immunoblot. To generate cells coexpressing VEGFR2Fas and FmsFas, a clone expressing VEGFR2Fas was transfected with LNCX/FmsFas virus and selected in G418 (500 μg/ml), and clones expressing FmsFas were identified by immunoblot. PAE cells expressing VEGFR2 or a FmsVEGFR2 chimeric receptor, which are used in five experiments as positive controls, were generated by the same retroviral transfection techniques.

Antibodies and Western Blot Analysis.

To demonstrate receptor expression, cells were lysed in lysis buffer (20 mm Tris, 137 mm NaCl, 10% glycerol, 1% Triton X-100, 0.15 units/ml aprotinin, 2 μm leupeptin, and 1 mm pefabloc), and equal amounts of lysate protein were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal anti-Fas Ab directed against the entire human Fas cytoplasmic domain (generated at BABCO), an anti-VEGFR2 Ab directed against the cytoplasmic domain (Santa Cruz Biotechnology), or an anti-HA Ab (Boehringer Mannheim). For caspase-3 cleavage experiments, cells in complete medium were treated with VEGF-A165, PlGF, or VEGF:PlGF (1 nm) for the indicated times in the absence or presence of cycloheximide (1 μg/ml in ethanol) or Z-VAD (40 μm in DMSO), lysed, and transferred as above, and Western blot analysis was performed with anticaspase-3 Ab (PharMingen) or anticleaved caspase-3 Ab (Cell Signaling Technology). Antiphosphotyrosine Ab was from Upstate Biotechnology. Blots were visualized by chemiluminescence (Amersham).

FACS Analysis of Receptor Expression.

Monoclonal rat Ab against mouse VEGFR-2 extracellular domain was purchased from PharMingen, and monoclonal rat Ab against human Fms receptor extracellular domain was purchased from Oncogene Research. FITC-conjugated goat-antirat Ab was obtained from ICN (Costa Mesa, CA). For fluorescence analysis, mAbs were used to stain cells at a concentration of 1 μg/106 cells. Routine analysis was performed by using a Becton Dickinson FACScan and Cell Quest software.

Mitogenesis Experiments.

Cells were plated in 24-well plates (5000 cells/well), quiesced overnight in F-12 medium with 0.5% serum, and stimulated for 24 h with VEGF-A165 (1 nm) or M-CSF (1 nm) or left unstimulated. Tritiated thymidine (0.5 μCi/well; NEN) was added at 20 h, and thymidine incorporation was assayed by scintillation counting of cell lysates at 24 h.

Experiments Using Anti-VEGFR2 Antibodies.

PAE cells expressing VEGFR2Fas or vector-transfected control cells were grown in six-well plates, and experiments were performed in complete medium with 10% serum. Cells were treated for 8 h with a primary Ab (either an anti-VEGFR2 Ab directed against the extracellular domain or a control Ab) or with no Ab. Primary anti-VEGFR2 antibodies were a rat monoclonal IgG2aκ anti-Flk1 Ab (0.5 μg/ml; PharMingen) or a goat polyclonal IgG anti-Flk1 Ab (1 μg/ml; R&D Systems). Control antibodies were isotype-matched IgG2aκ rat mAb (0.5 μg/ml; PharMingen) or goat IgG (1 μg/ml; Santa Cruz Biotechnology). To determine whether the addition of a secondary Ab might enhance the apoptotic response by clustering the primary Ab, goat antirat Ab (0.5 μg/ml; Oncogene Research) or mouse antigoat Ab (0.5 μg/ml; Santa Cruz Biotechnology) was added to some wells as indicated. Cycloheximide was used at 1 μg/ml.

Apoptosis Experiments.

PAE cells expressing empty vector, VEGFR2Fas, VEGFR2Fas(lprcg), or both VEGFR2Fas and FmsFas were stimulated with VEGF-A165 or M-CSF at the indicated concentrations and for the indicated times in complete F-12 medium with 10% calf serum. In some experiments, cells were also treated with cycloheximide (1 μg/ml in ethanol; Sigma), Z-VAD-fmk caspase inhibitor (40 μm in DMSO; BioMol), or vehicle (ethanol or DMSO). Cells were photographed after treatment with growth factors as indicated in each figure legend.

The chimeric VEGFR2Fas receptor cDNA was constructed by in-frame fusion of the complete extracellular and transmembrane domains of mouse VEGFR-2 (Flk1) with the complete cytoplasmic domain of human Fas receptor (Fig. 1,A). The VEGFR2Fas(lprcg) receptor is identical except for a C-to-G point mutation in the Fas death domain that inactivates apoptotic signaling (17). The FmsFas receptor was similarly constructed from the complete extracellular and transmembrane domains of human Fms receptor fused to the Fas cytoplasmic domain. A hemagglutinin tag was included on the carboxyl tail of each receptor. To investigate the function of VEGFR2Fas, VEGFR2Fas(lprcg), and FmsFas in endothelial cells, standard replication-defective retroviral techniques were used to generate stably transfected PAE cell lines expressing VEGFR2Fas and VEGFR2Fas(lprcg) or coexpressing VEGFR2Fas and FmsFas. PAE cells express low levels of endogenous VEGFRs (9, 18, 19, 20, 21) and have not been reported to be unusually sensitive to apoptotic signals or to be VEGF dependent. Expression of VEGFR2Fas in PAE cells was confirmed by Western blot analysis using an anti-Fas Ab directed against the cytoplasmic domain (Fig. 1,B). To compare FmsFas function to VEGFR2Fas function in the exact same cells, the VEGFR2Fas-expressing cells shown in Fig. 1,B were transfected with FmsFas virus, and stable double transfectants were selected. Coexpression of VEGFR2Fas and FmsFas is seen in Fig. 1,B. The expression level of FmsFas is higher than VEGFR2Fas in these cells. Receptors appear as doublets because of glycosylation of the extracellular domains, which occurs in native VEGFR2 and Fms receptors. VEGFR2Fas is larger than FmsFas because VEGFR2 has seven immunoglobulin-like extracellular domains, whereas Fms has only five. Both receptors migrated at their predicted sizes. FACS analysis using antibodies against the extracellular domains of VEGFR-2 or Fms receptor demonstrated surface expression of both VEGFR2Fas and FmsFas receptors (Fig. 1 C). Positive controls for FACS analysis included cell lines transfected with VEGFR-2 or a chimeric FmsVEGFR-2 receptor.

VEGFR2Fas Stimulates Endothelial Apoptosis in a VEGF Concentration- and Time-dependent Manner.

To determine whether VEGFR2Fas could induce endothelial apoptosis, VEGFR2Fas-expressing and vector-transfected control PAE cells were treated with VEGF (1 nm) in complete medium with 10% serum and examined at different time points for evidence of cell death. As seen in Fig. 2,A, VEGF treatment of vector-transfected control PAE cells had no effect, as would be expected, because PAE cells express low levels of endogenous VEGFRs and do not proliferate when stimulated with VEGF (19, 20), and the cells were already in 10% serum-containing medium. In stark contrast, VEGF rapidly stimulated apoptosis in VEGFR2Fas-expressing PAE cells. Beginning at 2 h, cells rounded up and detached from the plate. By 14 h, most cells had detached and fragmented. To examine the dose-response relationship, VEGFR2Fas-expressing cells were treated with escalating concentrations of VEGF in 10% serum-containing medium and examined for evidence of cell death at 14 h. As seen in Fig. 2,B, apoptotic changes were first apparent at 0.1 nm and were substantially greater at 1.0 nm, consistent with the known affinity of VEGF for VEGFR-2 (0.1–0.5 nm; Refs. 8 and 10). These results demonstrate that VEGFR2Fas stimulates apoptosis in a time- and VEGF concentration-dependent manner. Treatment with the VEGFR-1-specific ligand PlGF or the heterodimeric ligand VEGF:PlGF did not stimulate apoptosis (Fig. 2,B). VEGF:PlGF binds to VEGFR-2 (22), presumably via the VEGF chain. Unlike VEGF, neither VEGF:PlGF nor PlGF dimerized and autophosphorylated VEGFR-2 (Fig. 2 C). Among these three ligands, the ability to dimerize VEGFR2 correlated with stimulation of apoptosis by VEGFR2Fas.

Apoptosis induced by activation of Fas receptor has been intensively investigated and has well-described characteristics (12). These include formation of plasma membrane blebs, proteolytic activation of caspase-8 and caspase-3, cleavage of PARP, potentiation by treatment with cycloheximide, and inhibition by the caspase inhibitor Z-VAD-fmk (23). We examined whether apoptosis induced by VEGFR2Fas demonstrated these features of Fas apoptotic signaling, as might be predicted. Treatment of VEGFR2Fas-expressing cells with VEGF (1 nm for 5 h) stimulated typical apoptotic membrane blebbing (Fig. 3,A) not seen in control cells. Activation of Fas receptor initiates apoptotic signaling pathways that lead to the activation of the initiator caspase-8 and the effector caspase-3, which occur by cleavage of the caspase zymogen to generate active caspase (12). VEGF (1 nm) stimulated cleavage of caspase-8 in cells expressing VEGFR2Fas but not in vector control cells (Fig. 3,B, top panel), as well as PARP cleavage (Fig. 3,B, bottom panel). Similarly, treatment of VEGFR2Fas-expressing cells with VEGF (1 nm) stimulated the disappearance of the Mr 28,000 caspase-3 zymogen, which did not occur in control cells, and the formation of caspase-3 fragments, using an Ab specific for cleaved caspase-3 (Fig. 3,C). Neither PlGF nor VEGF:PlGF stimulated cleavage of caspase-3 (Fig. 3,D), consistent with the finding that neither growth factor stimulated apoptosis in VEGFR2Fas-expressing cells (Fig. 2,B). Fas-induced apoptosis is enhanced by treatment with cycloheximide and is inhibited by treatment with the caspase inhibitor Z-VAD-fmk (23). To determine whether apoptosis induced by VEGFR2Fas shared these features, VEGFR2Fas-expressing cells were treated with increasing concentrations of VEGF for 5 h in the presence of cycloheximide, ZVAD, or vehicle. VEGF treatment in the absence of cycloheximide or ZVAD induced membrane blebbing and cell fragmentation at 0.1–1 nm (Fig. 3,E, top panel). When cells were treated with both VEGF and cycloheximide, the apoptotic response was apparent at 10-fold lower concentrations of VEGF, beginning at 0.01 nm (Fig. 3,E, middle panel). In contrast, treatment with ZVAD inhibited VEGF-stimulated apoptosis, with minimal cell death even at 1 nm VEGF (Fig. 3,E, bottom panel). Similarly, cleavage of caspase-3 by VEGF treatment of VEGFR2Fas-expressing cells was enhanced by cycloheximide and inhibited by Z-VAD (Fig. 3 F). These results suggest that binding of VEGF to VEGFR2Fas activates caspase-mediated apoptotic signaling pathways in PAE cells similar to Fas-mediated apoptotic signals in hematopoietic cells.

A Functional Fas Death Domain Is Required for VEGFR2Fas-mediated Apoptosis.

To demonstrate that the Fas domain of VEGFR2Fas mediates apoptosis, an inactivating point mutation was introduced into VEGFR2Fas to create the receptor VEGFR2Fas(lprcg) (Fig. 1,A). The lprcg mutation is a point mutation in the Fas death domain that eliminates Fas apoptotic activity (17). PAE cells were generated that express VEGFR2Fas(lprcg) at levels similar to VEGFR2Fas-expressing cells (Fig. 4,A, using Ab against the HA tag on both receptors). As seen in Fig. 4,B, cells expressing VEGFR2Fas(lprcg) did not demonstrate VEGF-dependent apoptosis, which was seen in cells expressing VEGFR2Fas. Similarly, VEGFR2Fas(lprcg)-expressing cells did not demonstrate VEGF-dependent cleavage of caspase-3, whereas VEGFR2Fas-expressing cells did (Fig. 4 C). These results demonstrate that a functional Fas cytoplasmic death domain is required for the apoptotic activity of VEGFR2Fas.

Several lines of evidence indicate that the apoptotic activity of VEGFR2Fas is not attributable to dominant-negative interference with the functioning of endogenous VEGFRs: (a) as noted, VEGFR2Fas(lprcg) did not demonstrate any apoptotic activity, which it should have if VEGFR2Fas was functioning as a dominant-negative receptor; and (b) numerous investigators have reported that PAE cells express very low or undetectable levels of endogenous VEGFRs (9, 19, 21, 24). We have confirmed those findings. No expression of endogenous VEGFR2 was detectable in VEGFR2Fas-expressing cells or vector-transfected control cells using anti-VEGFR2 Ab (Fig. 5,A). The positive controls in this experiment are cell lines transfected with VEGFR2 or a FmsVEGFR2 chimeric receptor (Fig. 5,A, right two lanes). We further found no evidence of endogenous VEGFR tyrosine phosphorylation in VEGF-treated cells expressing VEGFR2Fas or vector-control cells (Fig. 5,B, left side). Positive controls for VEGFR autophosphorylation in this experiment included cells transfected with VEGFR2. We also found no evidence of any growth response after VEGF treatment in PAE cells (Fig. 5 C). The absence of endogenous VEGFR expression or activity in PAE cells, coupled with the absence of apoptotic activity by VEGFR2Fas(lprcg), demonstrates that the apoptotic activity of VEGFR2Fas is not attributable to a dominant-negative mechanism. Such a mechanism is additionally implausible because PAE cells do not require VEGF for survival, and, therefore, a dominant-negative VEGFR would not be expected to stimulate apoptosis.

Dimerization of VEGFR2Fas Is Sufficient to Stimulate Endothelial Apoptosis.

VEGF would be expected to homodimerize VEGFR2Fas as it does native VEGFR-2. To determine whether dimerization of VEGFR2Fas is sufficient to stimulate apoptosis, experiments were conducted using antibodies directed against the extracellular domain of VEGFR-2. Treatment of VEGFR2Fas-expressing cells with a rat monoclonal IgG that binds the extracellular domain of VEGFR-2 stimulated apoptosis (Fig. 6,A, C1 versus untreated control A1). The addition of a secondary goat antirat Ab to potentially enhance clustering did not significantly potentiate apoptosis (C2 versus C1), but the addition of cycloheximide did (C3 versus C1). Isotype matched rat mAb had no effect (D1-D4), and anti-VEGFR-2 mAb had no effect on vector-transfected control cells (Fig. 6,B). This result indicates that dimerization of VEGFR2Fas is sufficient to stimulate apoptosis but does not exclude the possibility that dimers cluster together to form higher-order oligomers. A goat polyclonal Ab against the extracellular domain of VEGFR-2 also stimulated apoptosis, and the activity was potentiated by cycloheximide (E1 and E3 versus A1). Control goat IgG had no apoptotic activity (F1–F4), and the polyclonal anti-VEGFR-2 Ab had no effect on vector-transfected control cells (Fig. 6 B). These results demonstrate again that dimerization or clustering of VEGFR2Fas is sufficient to stimulate apoptosis.

Dimerization of FmsFas Is Not Sufficient to Stimulate Endothelial Apoptosis.

It is not readily apparent why dimerization of the Fas cytoplasmic domain by VEGFR2Fas stimulates apoptosis in PAE cells, yet dimerization of the Fas cytoplasmic domain in other contexts does not, e.g., by anti-Fas antibodies or in the G-CSFRFas receptor (15, 25). Two trivial hypotheses would be that PAE cells are unusually sensitive to Fas apoptotic signaling or that the level of VEGFR2Fas expression is so high that simple dimerization is sufficient to initiate caspase activation. If either hypothesis was true, then a second dimerizing chimeric Fas receptor expressed at similar levels would be expected to induce apoptosis. To test this, cells expressing VEGFR2Fas were transfected with FmsFas, and a cell line was selected that coexpressed VEGFR2Fas and FmsFas. The level of FmsFas expression in these cells was greater than VEGFR2Fas (Fig. 1,B), yet treatment with the Fms ligand M-CSF did not simulate apoptosis, whereas treatment with VEGF did (Fig. 7). The inability of FmsFas to stimulate apoptosis is unlikely to result from failure to dimerize, because other Fms chimeric receptors are activated by M-CSF treatment (Fig. 5,B; Ref. 20). Nor is the lack of apoptosis attributable to signaling by endogenous Fms receptors, because we found no evidence that PAE control cells express endogenous Fms receptor or respond to M-CSF treatment (Fig. 5, B and C). The inability of FmsFas to stimulate apoptosis even when expressed at higher levels than VEGFR2Fas demonstrates that VEGFR2Fas-mediated apoptosis is not simply the result of high VEGFR2Fas expression levels or hypersensitivity to apoptosis signals in PAE cells.

We constructed a chimeric receptor composed of the extracellular, VEGF-binding domain of VEGFR-2 and the cytoplasmic domain of the Fas apoptosis receptor. When VEGFR2Fas was expressed in endothelial cells, treatment with VEGF stimulated apoptosis in a time- and VEGF concentration-dependent manner (Fig. 2). VEGFR2Fas-mediated apoptosis demonstrated features of Fas-mediated apoptosis, including membrane blebbing, activation of caspase-3, potentiation by cycloheximide, and inhibition by the caspase inhibitor ZVAD (Fig. 3). The apoptotic response was lost when the lprcg point mutation, which inactivates native Fas apoptotic signaling, was introduced into the Fas domain of VEGFR2Fas (Fig. 4). This observation, coupled with the fact that PAE cells express little or no endogenous VEGFRs, demonstrates that the apoptotic response is mediated by the Fas domain and not by a dominant-negative effect of VEGFR2Fas on endogenous VEGFRs. These results demonstrate that VEGFR2Fas functions as a VEGF-triggered endothelial apoptosis receptor by activating endogenous caspase-mediated apoptotic signaling pathways. Whereas other chimeric receptors that include components of Fas signaling have been shown to stimulate ligand- or Ab-dependent apoptosis (e.g., L-selectinFas, CD44Fas, FasFas-associated death domain, and FasCaspase8; Refs. 26, 27, 28, 29), this study appears to be the first demonstration that a growth factor can be switched to a death factor by a chimeric receptor designed to activate Fas apoptotic signaling.

Fas-mediated apoptosis is generally thought to require trimerization or oligomerization of Fas receptor by FasL (12). Evidence for this model comes from the observation that simple dimerization of Fas by anti-Fas Ab does not stimulate apoptosis, but cross-linking by secondary Ab or protein A does (15). In addition, a previously described chimeric receptor composed of the extracellular and transmembrane domains of the G-CSF receptor and the Fas cytoplasmic domain did not induce apoptosis, although it was dimerized by G-CSF ligand (25). However, other evidence suggests that dimerization of Fas cytoplasmic domains can be sufficient to induce apoptosis. Artificial dimerization of Fas cytoplasmic domains by chemical inducers of dimerization stimulated apoptosis, as did anti-CD44 monoclonal antibodies in cells expressing CD44Fas chimeric receptors (16, 26).

We found that VEGF, which binds to and dimerizes VEGFR-2 (Fig. 2,C), activated apoptotic signaling by VEGFR2Fas. In contrast, the heterodimeric ligand VEGF:PlGF, which binds to but did not dimerize VEGFR-2 (Fig. 2,C; Ref. 22), did not activate apoptotic signaling by VEGFR2Fas. This finding suggests that dimerization of VEGFR2Fas is necessary to stimulate apoptosis. We further found that a monoclonal IgG directed against the extracellular domain of VEGFR-2 stimulated apoptosis in cells expressing VEGFR2Fas (Fig. 6), indicating that dimerization of VEGFR2Fas is sufficient to initiate apoptotic signaling. Using a cell line coexpressing both chimeric receptors, we found that VEGFR2Fas, but not FmsFas, demonstrated ligand-dependent apoptosis (Fig. 7). Because both chimeric receptors would be expected to dimerize on ligand binding, it is not apparent why only VEGFR2Fas can activate apoptosis. This result suggests that the mechanism by which VEGFR2Fas stimulates apoptosis in PAE cells may be more complex than simple VEGFR2Fas dimerization, e.g., VEGF may generate VEGFR2Fas dimers that are highly stable or able to oligomerize, or VEGFR2Fas may form heteroreceptor complexes with endogenous VEGFRs (VEGFR-1, VEGFR-2, neuropilin-1, and neuropilin-2) that may be present at low levels in PAE cells that facilitate oligomerization of VEGFR2Fas and activation of caspases. The presence of the hemagglutinin tag on the C-tail of VEGFR2Fas may also enhance apoptosis by preventing the binding of FAP-1 to the extreme COOH-terminal amino acids of Fas, which normally inhibits Fas signaling (13). The failure of FmsFas, which also had a C-tail HA tag, to induce apoptosis suggests that blocking FAP-1 binding is not the primary basis for the apoptotic activity of VEGFR2Fas.

Apoptosis of VEGFR2Fas-expressing cells by VEGF is rapid, easily detectable, and can be enhanced by agents, such as cycloheximide, in contrast to the biological activities of VEGF on native endothelial cells, such as mitogenesis, which are typically modest and more time consuming to assay. Therefore, VEGFR2Fas-expressing cells may be a useful tool for identifying or confirming the activity of VEGF antagonists.

The ability of VEGFR2Fas to induce endothelial apoptosis suggests a novel approach to antiangiogenesis, in which a factor secreted by a tumor could be reprogrammed to function as an antiangiogenesis agent. If VEGFR2Fas were introduced into tumor vasculature and functioned in vivo as we have shown it does in vitro, VEGF produced by a tumor might induce apoptosis of its own vasculature. Because VEGF expression is up-regulated by ischemia, a potential unique advantage of such an approach is that tumor tissue made ischemic by induced vascular damage would likely increase secretion of VEGF, which would enhance the killing activity of VEGFR2Fas. The feasibility of this approach will require additional study.

Fig. 1.

Expression of chimeric VEGFR2Fas and FmsFas receptors in PAE cells. In A, the extracellular and transmembrane domains of mouse VEGFR-2 or human Fms receptor were fused with the entire cytoplasmic domain of the human Fas receptor. A hemagglutinin tag was included on the COOH terminus, not drawn to scale. In B, equal amounts of lysate protein from PAE cells stably transfected with vector, VEGFR2Fas, or VEGFR2Fas, plus FmsFas were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Ab against the Fas cytoplasmic domain. Receptors appear as doublets attributable to glycosylation of the extracellular domains. C, FACS analysis demonstrating surface expression of VEGFR2Fas and FmsFas. PAE cell lines stably expressing VEGFR2Fas + FmsFas, VEGFR2, or a chimeric FmsVEGFR2 receptor were incubated with antibodies against the extracellular domain of VEGFR2 or the extracellular domain of the Fms receptor, labeled with FITC-conjugated secondary antibodies, and analyzed by FACS. ····, fluorescence in the absence of Ab. Cells expressing VEGFR2 or FmsVEGFR2 serve as positive controls to demonstrate specificity of the antireceptor antibodies.

Fig. 1.

Expression of chimeric VEGFR2Fas and FmsFas receptors in PAE cells. In A, the extracellular and transmembrane domains of mouse VEGFR-2 or human Fms receptor were fused with the entire cytoplasmic domain of the human Fas receptor. A hemagglutinin tag was included on the COOH terminus, not drawn to scale. In B, equal amounts of lysate protein from PAE cells stably transfected with vector, VEGFR2Fas, or VEGFR2Fas, plus FmsFas were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Ab against the Fas cytoplasmic domain. Receptors appear as doublets attributable to glycosylation of the extracellular domains. C, FACS analysis demonstrating surface expression of VEGFR2Fas and FmsFas. PAE cell lines stably expressing VEGFR2Fas + FmsFas, VEGFR2, or a chimeric FmsVEGFR2 receptor were incubated with antibodies against the extracellular domain of VEGFR2 or the extracellular domain of the Fms receptor, labeled with FITC-conjugated secondary antibodies, and analyzed by FACS. ····, fluorescence in the absence of Ab. Cells expressing VEGFR2 or FmsVEGFR2 serve as positive controls to demonstrate specificity of the antireceptor antibodies.

Close modal
Fig. 2.

VEGFR2Fas stimulates endothelial apoptosis in a time-dependent and VEGF concentration-dependent manner. In A, PAE cells expressing empty vector or VEGFR2Fas were stimulated with VEGF-165 (1 nm) in 10% serum for times as indicated. Apoptosis is first evident at 2 h and is extensive by 14 h. In B, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165 (0.01–1 nm) in 10% serum for 12 h or with PlGF (1 nm) or VEGF:PlGF (1 nm). PlGF and VEGF:PlGF do not stimulate apoptosis. In C, PAE cells stably expressing VEGFR2 were stimulated with VEGF165, PlGF, or VEGF:PlGF (1 nm) for 5 min. Lysates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine Ab, demonstrating that PlGF and VEGF:PlGF do not stimulate autophosphorylation of VEGFR2.

Fig. 2.

VEGFR2Fas stimulates endothelial apoptosis in a time-dependent and VEGF concentration-dependent manner. In A, PAE cells expressing empty vector or VEGFR2Fas were stimulated with VEGF-165 (1 nm) in 10% serum for times as indicated. Apoptosis is first evident at 2 h and is extensive by 14 h. In B, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165 (0.01–1 nm) in 10% serum for 12 h or with PlGF (1 nm) or VEGF:PlGF (1 nm). PlGF and VEGF:PlGF do not stimulate apoptosis. In C, PAE cells stably expressing VEGFR2 were stimulated with VEGF165, PlGF, or VEGF:PlGF (1 nm) for 5 min. Lysates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine Ab, demonstrating that PlGF and VEGF:PlGF do not stimulate autophosphorylation of VEGFR2.

Close modal
Fig. 3.

VEGFR2Fas-mediated apoptosis displays features characteristic of Fas-mediated apoptosis. In A, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165 (1 nm) in medium with 10% serum for 5 h. Membrane blebbing is indicated by arrowheads. In B, PAE cells expressing VEGFR2Fas or empty vector were stimulated with VEGF (1 nm) in medium with 10% serum for the indicated times. Lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-8 Ab or anti-PARP Ab. In C, PAE cells expressing VEGFR2Fas or empty vector were stimulated with VEGF (1 nm) in medium with 10% serum for the indicated times. Lysates were separated by SDS-PAGE and immunoblotted with anticaspase-3 Ab or anticleaved caspase-3 Ab. In D, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165, PlGF, or VEGF:PlGF (1 nm) for times as indicated. Cell lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-3 Ab. In E, PAE cells expressing VEGFR2Fas were stimulated for 12 h with increasing concentrations of VEGF in the presence of cycloheximide (1 μg/ml), Z-VAD-fmk (40 μm), or vehicle. In F, cells expressing VEGFR2Fas or vector were stimulated with VEGF-165 (1 nm) in the absence or presence of cycloheximide (1 μg/ml) or Z-VAD (40 μm). Cell lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-3 Ab, demonstrating that CHX enhances and Z-VED inhibits caspase-3 cleavage.

Fig. 3.

VEGFR2Fas-mediated apoptosis displays features characteristic of Fas-mediated apoptosis. In A, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165 (1 nm) in medium with 10% serum for 5 h. Membrane blebbing is indicated by arrowheads. In B, PAE cells expressing VEGFR2Fas or empty vector were stimulated with VEGF (1 nm) in medium with 10% serum for the indicated times. Lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-8 Ab or anti-PARP Ab. In C, PAE cells expressing VEGFR2Fas or empty vector were stimulated with VEGF (1 nm) in medium with 10% serum for the indicated times. Lysates were separated by SDS-PAGE and immunoblotted with anticaspase-3 Ab or anticleaved caspase-3 Ab. In D, PAE cells expressing VEGFR2Fas were stimulated with VEGF-165, PlGF, or VEGF:PlGF (1 nm) for times as indicated. Cell lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-3 Ab. In E, PAE cells expressing VEGFR2Fas were stimulated for 12 h with increasing concentrations of VEGF in the presence of cycloheximide (1 μg/ml), Z-VAD-fmk (40 μm), or vehicle. In F, cells expressing VEGFR2Fas or vector were stimulated with VEGF-165 (1 nm) in the absence or presence of cycloheximide (1 μg/ml) or Z-VAD (40 μm). Cell lysates were separated by SDS-PAGE and immunoblotted with anticleaved caspase-3 Ab, demonstrating that CHX enhances and Z-VED inhibits caspase-3 cleavage.

Close modal
Fig. 4.

An inactivating point mutation in the Fas death domain abolishes VEGFR2Fas-mediated apoptosis. In A, equal amounts of lysate protein from cells expressing vector, VEGFR2Fas, or VEGFR2Fas(lprcg), which contains an inactivating point mutation in the Fas death domain, were electrophoresed and immunoblotted with antihemagglutinin Ab. In B, PAE cells expressing VEGFR2Fas or VEGFR2Fas(lprcg) were stimulated with VEGF-165 (1 nm) for times as indicated. Cells expressing VEGFR2Fas(lprcg) do not undergo apoptosis. In C, PAE cells expressing VEGFR2Fas or VEGFR2Fas(lprcg) were stimulated with VEGF-165 (1 nm) for times as indicated, and lysates were immunoblotted with anticaspase-3 and anticleaved caspase-3 antibodies. Cleavage of caspase-3 was not seen in cells expressing VEGFR2Fas(lprcg).

Fig. 4.

An inactivating point mutation in the Fas death domain abolishes VEGFR2Fas-mediated apoptosis. In A, equal amounts of lysate protein from cells expressing vector, VEGFR2Fas, or VEGFR2Fas(lprcg), which contains an inactivating point mutation in the Fas death domain, were electrophoresed and immunoblotted with antihemagglutinin Ab. In B, PAE cells expressing VEGFR2Fas or VEGFR2Fas(lprcg) were stimulated with VEGF-165 (1 nm) for times as indicated. Cells expressing VEGFR2Fas(lprcg) do not undergo apoptosis. In C, PAE cells expressing VEGFR2Fas or VEGFR2Fas(lprcg) were stimulated with VEGF-165 (1 nm) for times as indicated, and lysates were immunoblotted with anticaspase-3 and anticleaved caspase-3 antibodies. Cleavage of caspase-3 was not seen in cells expressing VEGFR2Fas(lprcg).

Close modal
Fig. 5.

PAE cells do not express significant levels of endogenous VEGFR2 or Fms receptors. In A, equal amounts of lysate protein from cells stably transfected with vector, VEGFR2Fas, VEGFR2, or FmsVEGFR2 were immunoblotted with Ab against the cytoplasmic domain of VEGFR2. No detectable VEGFR2 is expressed in vector- or VEGFR2Fas-transfected cells. In B, PAE cells expressing vector, VEGFR2Fas, VEGFR2, or FmsVEGFR2 were quiesced and stimulated with VEGF-165 (V) or M-CSF (C; 1 nm) for 5 min. Cell lysates were immunoblotted with antiphosphotyrosine Ab, demonstrating that VEGF and M-CSF do not generate any endogenous receptor autophosphorylation in vector- or VEGFR2Fas-transfected cells. In C, PAE cells expressing vector were quiesced and stimulated for 24 h with VEGF-165 (V) or M-CSF (C; 1 nm). Thymidine incorporation in the last 2 h was assayed, demonstrating that VEGF and M-CSF do not stimulate any growth response in vector-transfected PAE cells.

Fig. 5.

PAE cells do not express significant levels of endogenous VEGFR2 or Fms receptors. In A, equal amounts of lysate protein from cells stably transfected with vector, VEGFR2Fas, VEGFR2, or FmsVEGFR2 were immunoblotted with Ab against the cytoplasmic domain of VEGFR2. No detectable VEGFR2 is expressed in vector- or VEGFR2Fas-transfected cells. In B, PAE cells expressing vector, VEGFR2Fas, VEGFR2, or FmsVEGFR2 were quiesced and stimulated with VEGF-165 (V) or M-CSF (C; 1 nm) for 5 min. Cell lysates were immunoblotted with antiphosphotyrosine Ab, demonstrating that VEGF and M-CSF do not generate any endogenous receptor autophosphorylation in vector- or VEGFR2Fas-transfected cells. In C, PAE cells expressing vector were quiesced and stimulated for 24 h with VEGF-165 (V) or M-CSF (C; 1 nm). Thymidine incorporation in the last 2 h was assayed, demonstrating that VEGF and M-CSF do not stimulate any growth response in vector-transfected PAE cells.

Close modal
Fig. 6.

Antibodies against VEGFR2 extracellular domain stimulate VEGFR2Fas-mediated apoptosis. In A, to determine whether antibodies directed against the extracellular domain of VEGFR2 stimulate apoptosis in cells expressing VEGFR2Fas, cells growing in six-well plates in complete medium were treated for 8 h with a primary Ab, either rat monoclonal anti-VEGFR2 Ab (0.5 μg/ml; C1–C4) or goat polyclonal anti-VEGFR2 Ab (1 μg/ml; E1–E4). Control primary antibodies were rat isotype Ab (0.5 μg/ml; D1–D4) or goat IgG (1 μg/ml; F1–F4). Cells in column 2 were treated with a secondary Ab to induce clustering, either goat antirat (0.5 μg/ml) or mouse antigoat (0.5 μg/ml). Cells in column 3 were treated with cycloheximide (CHX; 1 μg/ml) or ethanol vehicle. Cells in column 4 were treated with both secondary Ab and CHX. Negative controls included untreated cells (A1) and cells treated with secondary Ab and/or CHX but without primary Ab (A2–A4 and B2–B4). VEGF-treated cells (1 nm; B1) are the positive control. Both anti-VEGFR2 antibodies stimulated apoptosis (C1 and E1 versus A1), which was not seen in cells treated with control antibodies (D1 or F1). The addition of a secondary Ab did not significantly enhance killing (C2 versus C1 and E2 versus E1), but the addition of cycloheximide to the anti-VEGFR2 antibodies significantly enhanced apoptosis (C3 versus C1 and E3 versus E1). Cycloheximide alone or in combination with control antibodies did not stimulate apoptosis (A3, A4, B4, D3, D4, F3, and F4). Vector-transfected cells did not demonstrate apoptosis under any of these conditions (6B). Stimulation of apoptosis by an IgG mAb directed against the extracellular domain of VEGFR2 (C1–C4) indicates that dimerization of VEGFR2Fas is sufficient to stimulate apoptosis.

Fig. 6.

Antibodies against VEGFR2 extracellular domain stimulate VEGFR2Fas-mediated apoptosis. In A, to determine whether antibodies directed against the extracellular domain of VEGFR2 stimulate apoptosis in cells expressing VEGFR2Fas, cells growing in six-well plates in complete medium were treated for 8 h with a primary Ab, either rat monoclonal anti-VEGFR2 Ab (0.5 μg/ml; C1–C4) or goat polyclonal anti-VEGFR2 Ab (1 μg/ml; E1–E4). Control primary antibodies were rat isotype Ab (0.5 μg/ml; D1–D4) or goat IgG (1 μg/ml; F1–F4). Cells in column 2 were treated with a secondary Ab to induce clustering, either goat antirat (0.5 μg/ml) or mouse antigoat (0.5 μg/ml). Cells in column 3 were treated with cycloheximide (CHX; 1 μg/ml) or ethanol vehicle. Cells in column 4 were treated with both secondary Ab and CHX. Negative controls included untreated cells (A1) and cells treated with secondary Ab and/or CHX but without primary Ab (A2–A4 and B2–B4). VEGF-treated cells (1 nm; B1) are the positive control. Both anti-VEGFR2 antibodies stimulated apoptosis (C1 and E1 versus A1), which was not seen in cells treated with control antibodies (D1 or F1). The addition of a secondary Ab did not significantly enhance killing (C2 versus C1 and E2 versus E1), but the addition of cycloheximide to the anti-VEGFR2 antibodies significantly enhanced apoptosis (C3 versus C1 and E3 versus E1). Cycloheximide alone or in combination with control antibodies did not stimulate apoptosis (A3, A4, B4, D3, D4, F3, and F4). Vector-transfected cells did not demonstrate apoptosis under any of these conditions (6B). Stimulation of apoptosis by an IgG mAb directed against the extracellular domain of VEGFR2 (C1–C4) indicates that dimerization of VEGFR2Fas is sufficient to stimulate apoptosis.

Close modal
Fig. 7.

FmsFas does not stimulate apoptosis. PAE cells coexpressing VEGFR2Fas and FmsFas were stimulated with VEGF (1 nm) or M-CSF (1 nm) for 6–48 h in medium with 10% serum. M-CSF does not stimulate apoptosis, whereas VEGF does.

Fig. 7.

FmsFas does not stimulate apoptosis. PAE cells coexpressing VEGFR2Fas and FmsFas were stimulated with VEGF (1 nm) or M-CSF (1 nm) for 6–48 h in medium with 10% serum. M-CSF does not stimulate apoptosis, whereas VEGF does.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by grants from the American Cancer Society (to T. P. Q. and M. C. N.), the French Foundation for Medical Research and Education (to T. P. Q.), Daiichi Ltd. (to L. T. W.), and a VA Career Development Award (to M. C. N.).

8

The abbreviations used are: VEGF, vascular endothelial growth factor; FAP, Fas-associated phosphatase; PAE, porcine aortic endothelial; M-CSF, macrophage colony stimulating factor; G-CSF, granulocyte colony stimulating factor; PlGF, placenta growth factor; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; Ab, antibody; mAb, monoclonal antibody; PARP, poly(ADP-ribose) polymerase.

We thank Drs. Shigekazu Nagata for human Fas cDNA and James Torchia for reading the manuscript.

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