Research Article
`
`Antagonism of Sphingosine-1-Phosphate Receptors by FTY720
`Inhibits Angiogenesis and Tumor Vascularization
`
`Kenneth LaMontagne,1 Amanda Littlewood-Evans,2 Christian Schnell,2 Terence O’Reilly,2
`Lorenza Wyder,2 Teresa Sanchez,3 Beatrice Probst,2 Jeannene Butler,1 Alexander Wood,4
`Gene Liau,4 Eric Billy,2 Andreas Theuer,2 Timothy Hla,3 and Jeanette Wood2
`
`1Novartis Institutes for BioMedical Research, East Hanover, New Jersey; 2Novartis Institutes for BioMedical Research, Basel, Switzerland;
`3University of Connecticut Health Center, Farmington, Connecticut; and 4Novartis Institutes for BioMedical Research, Inc.,
`Cambridge, Massachusetts
`
`Abstract
`FTY720, a potent immunomodulator, becomes phosphorylated
`in vivo (FTY-P) and interacts with sphingosine-1-phosphate
`(S1P) receptors. Recent studies showed that FTY-P affects
`vascular endothelial growth factor (VEGF)–induced vascular
`permeability, an important aspect of angiogenesis. We show
`here that FTY720 has antiangiogenic activity, potently abro-
`gating VEGF- and S1P-induced angiogenesis in vivo in growth
`factor implant and corneal models. FTY720 administration
`tended to inhibit primary and significantly inhibited meta-
`static tumor growth in a mouse model of melanoma growth. In
`combination with a VEGFR tyrosine kinase inhibitor PTK787/
`ZK222584, FTY720 showed some additional benefit. FTY720
`markedly inhibited tumor-associated angiogenesis, and this
`was accompanied by decreased tumor cell proliferation and
`increased apoptosis. In transfected HEK293 cells, FTY-P
`internalized S1P1 receptors, inhibited their recycling to the
`cell surface, and desensitized S1P receptor function. Both
`FTY720 and FTY-P apparently failed to impede VEGF-produced
`increases in mitogen-activated protein kinase activity in
`human umbilical vascular endothelial cells (HUVEC), and
`unlike its activity in causing S1PR internalization, FTY-P did
`not result in a decrease of surface VEGFR2 levels in HUVEC
`cells. Pretreatment with FTY720 or FTY-P prevented S1P-
`induced Ca2+ mobilization and migration in vascular endo-
`thelial cells. These data show that functional antagonism of
`vascular S1P receptors by FTY720 potently inhibits angiogen-
`esis; therefore, this may provide a novel therapeutic approach
`for pathologic conditions with dysregulated angiogenesis.
`(Cancer Res 2006; 66(1): 221-31)
`
`Introduction
`
`Angiogenesis, the formation of new blood vessels from preexist-
`ing vessels,
`is a normal aspect of the physiologic remodeling
`processes that occurs in wound healing and during the female
`reproductive cycle. However,
`in pathologic situations, such as
`rheumatoid arthritis, diabetic retinopathy, and tumor development,
`abnormally enhanced neovascularization is a major contributory
`
`Note: K. LaMontagne and A. Littlewood-Evans contributed equally to this work.
`K. LaMontagne and J. Butler are currently at J&J PRD, Raritan, NJ (klamontagne@
`prdus.jnj.com).
`PTK787/ZK222584 is a co-development compound by Novartis AG and Schering AG.
`Requests for reprints: Amanda Littlewood-Evans, Novartis NIBR AG, K125.1.20,
`Klybeck Strasse, Basel, CH4002, Switzerland. Phone: 41-61-696-1023; Fax: 41-61-696-
`6242; E-mail: amanda.littlewood-evans@novartis.com.
`I2006 American Association for Cancer Research.
`doi:10.1158/0008-5472.CAN-05-2001
`
`factor for disease progression (1, 2). The initiation of pathology-
`associated angiogenesis involves vascular permeability changes,
`driven by angiogenic factors, such as vascular endothelial growth
`factor (VEGF; ref. 3). This leads to fibrin deposition, plasmin
`activation, basement membrane degradation, and ultimately
`endothelial cell migration and proliferation, recruitment of mural
`cells, and vessel maturation (4).
`Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metab-
`olite secreted by platelets upon activation, is a potent proangio-
`genic molecule, which acts by binding various members of the
`G-protein–coupled receptor (GPCR) family of S1P receptors (S1P-R;
`refs. 5, 6). A novel immunosuppressant agent currently in clinical
`trials for renal transplant rejection (FTY720) and its metabolite of
`cellular kinase(s) FTY720 phosphate (FTY-P; ref. 7) bear structural
`similarity to sphingosine and S1P, respectively. FTY-P binds at low
`nanomolar concentrations to four of five S1P-Rs, S1P1, S1P3, S1P4,
`and S1P5 (8). Recently, we have shown that FTY-P can act in a
`similar manner to S1P, stimulating endothelial cell signaling,
`migration, survival, and differentiation (9). By recruiting adherens
`junction proteins to the endothelial cell-cell junctions (10), FTY720
`has also been shown to antagonize VEGF-induced permeability
`of blood vessels (10, 11). Tumor-associated blood vessels are
`permeable and elicit
`tissue extracellular fluid extravasation;
`therefore, this prompted us to investigate whether FTY720 exerts
`any antiangiogenic and antitumor activity in vivo by affecting
`vessel permeability.
`In this report, we show that FTY720 at clinically relevant doses,
`inhibits both S1P- and VEGF-induced angiogenesis, and impedes
`primary and metastatic tumor growth in a murine model of
`melanoma. Additionally, combination of FTY720 with the VEGFR
`tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK)
`further
`reduces the growth of the tumors and metastases. These findings
`suggest that targeting S1P receptors may provide a novel ther-
`apeutic approach in cancer treatment.
`
`Materials and Methods
`
`Materials. S1P was purchased from BioMol Research Labs,
`Inc.
`(Plymouth Meeting, PA). FTY720 and all other related compounds
`mentioned herein were generously provided by the Novartis Transplanta-
`tion Group (Basel, Switzerland) and prepared as described previously (9).
`Human umbilical vein endothelial cells (HUVEC), from Vec Technologies,
`Inc. (Rensselaer, NY), were maintained in MCDB 131 Complete media
`(Vec Technologies) and were used from passages 4 to 7.
`Female C57/Bl6 mice were obtained from The Jackson Laboratory
`(Bar Harbor, ME) or IFFA Credo (L’ Arbresle, France). Female mice (MAG
`and NIH/Tif), weighing 18 to 20 g (6-8 weeks old), were obtained from the
`Novartis animal breeding facility. All animal experiments done in Switzer-
`land were done in strict adherence to the Swiss law for animal protection,
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`and experiments done in The United States were conducted in accordance
`with the Novartis Animal Care and Use Committee.
`The S1P analogues FTY720, NVP-AAL149, and NVP-AAL151 and the
`VEGFR receptor tyrosine kinase inhibitor NVP-AAL993 were synthesized by
`Novartis AG (Basel, Switzerland). The VEGFR receptor tyrosine kinase
`inhibitor PTK/ZK was synthesized by Schering AG (Berlin, Germany) in
`collaboration with Novartis.
`Fluorescence imaging plate reader Ca2+ mobilization assay.
`Fluorescence imaging plate reader (FLIPR) assay with HUVECs was carried
`out as described previously (9). Briefly,
`titrated compounds were
`preincubated with cells in a 96-well plate for 3 hours. Subsequently, the
`cell plates and ligand plates (containing 500 nmol/L S1P final concentra-
`tion) were loaded into the FLIPR. The inhibition of S1P-induced calcium
`mobilization by FTY720, FTY-P, and NVP-AAL151 was plotted with EXCEL
`and SigmaPlot, and IC50 was determined for each compound.
`Migration assay. The 4-hour migration assay was carried out using the
`BD Biocoat FluoroBlok System as described previously (9). HUVECs were
`preincubated for 30 minutes with compounds in MCDB 131 basal media
`(Vec Technologies), containing 0.1% bovine serum albumin (BSA, delipi-
`dized, BD Biosciences). S1P was diluted in the same media to a final
`concentration of 500 nmol/L and added to the bottoms of the assay plate
`wells. Migrated cells were stained with Calcein AM (Molecular Probes,
`Eugene, OR) and quantified with a CytoFluor II (PerSeptive Biosystems,
`Framingham, ME) fluorescent plate reader.
`Mouse corneal micropocket assay. This method has been previously
`described in detail (12). Briefly, pellets containing the slow-release polymer
`Hydron and sucralfate with 180 ng rHuVEGF165 were implanted into the
`cornea of female C57BL/6J mice. Daily oral treatment with FTY720 (0.3 or
`3 mg/kg) or vehicle (10 mL/kg, 5% w/v glucose) was started 24 hours later.
`The eyes were routinely examined by slit-lamp biomicroscopy (Nikon
`FS-3V), and on day 6, mice were sacrificed, and the vascular response was
`quantified using a linear reticule through the slit lamp. Inhibition was
`determined by the formula 0.2p  new blood vessel length  clock hours of
`neovessels. The circumferential zone was measured as clock hours with a
`360-degree reticule (where 30 degrees of arc equals 1 clock hour). The data
`are reported as the % inhibition of blood vessel growth compared with
`the vehicle group.
`Chamber assay (S1P and VEGF dependent). This assay has been
`described previously (13). Briefly, sterile porous Teflon chambers were filled
`with 0.8% agar (BBL Nr. 11849, Becton Dickinson, Meylan, France)
`containing heparin (20 units/mL) with or without growth factors: VEGF165
`(2 Ag/mL; Tumor Center, Freiburg, Germany), or 5 Amol/L S1P (ANAWA
`Biomedical, Zu¨rich, Switzerland). The chamber was implanted s.c. on the
`dorsal flank of female mice (MAG and NIH/Tif). Animals were treated with
`FTY720 (0.3 or 3 mg/kg orally), NVP-AAL151 or NVP-AAL149 (2.5 mg/kg i.v.)
`or NVP-AAL993 (100 mg/kg orally) 4 to 6 hours before chamber
`implantation and then once daily for a further 3 days. On the fourth day
`after implantation, animals were sacrificed, and the vascularized fibrous
`tissue formed around each implant carefully removed and weighed. Tissue
`samples were then homogenized in 1 mL of radioimmunoprecipitation
`assay (RIPA) buffer [50 mmol/L Tris-HCl (pH 7.2), 120 mmol/L NaCl,
`1 mmol/L EDTA, 6 mmol/L EGTA, 1% (v/v) NP40, 20 mmol/L NaF, to which
`fresh 1 mmol/L phenylmethylsulfonyl fluoride and 1 mmol/L Na-vanadate
`were added], centrifuged, and filtered. The amount of hemoglobin present
`in the supernatant was determined by spectrophotometric analysis at 540
`nm using the Drabkin reagent kit (Sigma hemoglobin #525, Sigma Chemical
`Co., Ltd., Poole, Dorset, England).
`Tie-2 measurements. Tie-2 levels were determined using an ELISA
`method. Nunc (Naperville, IL) Maxsisorb 96-well plates were coated over
`night at 4jC with the capture antibody, anti-Tie-2 AB33 (UBI, Hauppauge,
`NY), with a concentration of 2 Ag/mL (100 AL/well). Wells were washed in
`TPBS (Tween 80 PBS) and blocked by incubating with 3% Top-Block (Juro,
`Lucerne, Switzerland) for 2 hours at room temperature. After washing,
`300 Ag of protein lysates were incubated for 2 hours before further washing
`and addition of a complex of detection antibody, goat anti-mouse Tie-2
`(R&D Systems, Minneapolis, MN; 0.5 Ag/mL) and alkaline phosphate
`conjugated to monoclonal antibody (mAb) anti-goat (Sigma, St. Louis, MO;
`
`diluted 1:6,000) in TPBS + 0.1%Top-Block for 1 hour at room temperature.
`After washing, Tie-2 antibody complexes were detected by incubating with
`p-nitrophenyl phosphate (Sigma, tablets) and reading absorbance with an
`ELISA reader at 405 nm.
`Recombinant human extracellular domain of Tie-2 fused to the constant
`regions of human IgG1 (sTie-2Fc) dissolved in RIPA buffer was used as
`standard in a concentration range from 0.1 to 300 ng/well (Tie-2Fc was a
`kind gift from Georg Martiny-Baron, Novartis).
`VEGF-induced microvascular permeability. Heparin immobilized
`acrylic beads (50-100 Am in diameter) were incubated overnight with
`PBS/O or a solution of 1 Ag VEGF in 10 AL PBS/O. Subsequently, the beads
`were implanted s.c. in both ears of female MAG mice (8-10 beads per ear).
`Vascular permeability of the newly formed vessels was visualized after
`2 days using Evans blue dye (2%, 10 ml/kg) that was injected i.v. 5 minutes
`before sacrifice. Measurements of the dye extravasation area (mm2) were
`carried out using pixel-based threshold in a computer-assisted image
`analysis software (KS-400 3.0 imaging system, Zeiss, Jena, Germany). Mice
`were treated with FTY720 (0.3 and 3 mg/kg orally) or PTK/ZK (100 mg/kg
`orally) 2 hours before Evans blue injection.
`Tumor model. The syngeneic B16/BL6 murine melanoma model,
`previously shown to be responsive to antiangiogenic therapy (13), was
`used to evaluate the antitumor activity of FTY720. B16BL6 melanoma cells
`(kind gift from Dr. Isaiah J. Fidler, Texas Medical Center, Houston, TX) were
`cultured until confluency. Tumor cells (1 AL, 5  104/AL) was injected
`intradermally into the dorsal pinna of both ears of syngeneic female C57BL/
`6 mice. Measurements of primary tumor area (mm2) were carried out on
`days 7, 14, and 21 after tumor cell inoculation using computer-assisted
`image analysis software (KS-400 3.0 imaging system, Zeiss) and a specially
`designed macro. From days 7 to 21, mice were treated orally once daily with
`either vehicle PEG300 (5 mL/kg), FTY720 (3 mg/kg), PTK/ZK (100 mg/kg),
`or a combination of the two compounds at the above doses. Mice were
`sacrificed on day 21, and cranial lymph node metastases were weighed and
`then frozen in ornithine carbamyl transferase cryofreezing medium for
`histologic analysis.
`Histologic analysis and lectin perfusion. Lectin (200 AL) from Ricinus
`communis agglutinin-1, FITC conjugated (Vector Labs, Burlingame, CA),
`at a concentration of 1 Ag/AL in sterile 0.9% NaCl was injected into the tail
`vein of a B16/BL6 melanoma bearing C57/B6J mouse. The mouse was left
`for 30 minutes before sacrifice and tumor excision. Frozen sections (12 Am)
`were subjected to immunohistochemical analysis as described previously
`(14). Antibodies used were rat anti-mouse CD31 (BD PharMingen, San Diego,
`CA; diluted 1:600 in PBS), rabbit anti-mouse active caspase 3 (Oncogene,
`Uniondale, NY; diluted 1:10 in PBS), and rabbit anti mouse Ki67 (Neo-
`markers, Fremont, CA; diluted 1:200 in PBS). Secondary antibodies were goat
`anti-rabbit or goat anti-rat ALEXA 568 or 488 (Molecular Probes, 1:400).
`Fluorescence-activated cell sorting analysis. Mice bearing B16/BL6
`melanoma tumors were treated daily for 7 days (days 7-14) with FTY720
`(3 mg/kg orally), PTK/ZK (100 mg/kg orally) or the combination of both
`drugs. At the end of treatment, mice were sacrificed, cranial lymph node
`metastases were surgically removed and minced into small pieces, and a
`single-cell suspension was formed by collagenase/dispase treatment and
`subjected to fluorescence-activated cell sorting (FACS) analysis. Due to their
`small size, tumor cells were detected as a distinct population in the scatter
`plot and can be accordingly gated and enumerated.
`HUVEC cells were treated with vehicle alone or FTY-P (10 and 100 nmol/L)
`for 30 minutes and were analyzed by FACS for VEGFR2 levels on the cell
`surface. Cells were trypsinized, washed twice with PBS containing 10% (v/v)
`fetal bovine serum (FBS), and incubated 10 minutes on ice before the
`addition of mouse mAb anti-VEGFR2/KDR 1495.12.14 (13) antibody
`developed within our laboratories in Novartis (2 Ag mAb/106 cells). After
`1 hour of incubation on ice, cells were washed twice in PBS plus 10% FBS,
`and RPE-labeled anti-mouse was added to the cells (BD PharMingen). FACS
`acquisition and analysis were done on a FACSCalibur using Cell Quest
`Software (Becton Dickinson).
`Analysis of S1P1 receptor localization. HEK293 cells expressing S1P1-
`GFP fusion protein (15) were used in this assay. Cells were serum starved
`for 2 hours and treated with indicated concentrations of S1P or FTYP or
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`structural analogues as described (10). At specific times, cells were fixed and
`imaged on a Zeiss 510 confocal microscope as previously described (15).
`Blood cell counts. Blood analysis was done using a commercially
`available blood analyzer ABC VET 16 (Axon Lab AG, Baden-Da¨ttwil,
`Switzerland). Blood was collected in EDTA tubes via the vena cava inferior
`immediately after sacrifice of the mice by CO2 inhalation. An aliquot (12 AL)
`of EDTA-blood obtained from all groups of mice treated for 21 days was
`taken 24 hours after the last dose and analyzed for WBC, RBC, platelets,
`lymphocytes, monocytes, and granulocytes.
`Proliferation assay. Subconfluent B16/BL6 melanoma cells were seeded
`at a density of 3  103 per well into 96-well plates and incubated at 37jC
`and 5% CO2 in growth medium (10% FBS in MEM EBS, Amimed). After
`another 24 hours, the medium was renewed and PTK787/ZK222584,
`FTY720, FTY-P, or vehicle were added. After 8 hours of
`incubation,
`bromodeoxyuridine (BrdUrd) labeling solution was added, and cells were
`incubated a further 16 hours before fixation, blocking, and addition of
`peroxidase-labeled anti-BrdUrd antibody. Bound antibody was then
`detected using 3,3V5,5V-tetramethylbenzidine substrate, which forms a
`colored reaction product that is quantified spectrophotometrically at
`450 nm.
`Apoptosis assay. Subconfluent B16/BL6 melanoma cells were seeded at
`a density of 3  103 per well into 96-well plates and incubated at 37jC and
`5% CO2 in growth medium (10% FCS in MEM EBS, Amimed). After another
`24 hours, the medium was renewed and PTK787/ZK222584, FTY720,
`FTY-P, or vehicle was added. Twenty-four hours later, induced cell death
`was measured photometrically through determination of cytoplasmic
`histone-associated DNA fragments, upon instructions of the supplier from
`the kit (Cell Death Detection ELISAPLUS, Roche, Indianapolis, IN).
`Western analysis. HUVECs at 80% confluency in EBM medium
`containing 0.5% (v/v) FBS were incubated with 500 nmol/L of either
`FTY720, FTY-P, or PTK/ZK for either 20 minutes or 1 hour. For the last
`10 minutes of the incubation time, some of the HUVECs were stimulated
`with VEGF 10 ng/mL, and cells were subsequently lysed with RIPA buffer
`[50 mmol/L Tris-HCl (pH 7.2), 120 mmol/L NaCl, 1 mmol/L EDTA (pH 8),
`6 mmol/L EGTA (pH 8.5), 1% (v/v) NP40, and 20 mmol/L NaF]. Ten
`micrograms of lysates were run on a 10% SDS gel and blotted. The gel was
`first probed with p44/42 mitogen-activated protein kinase (MAPK) antibody
`(Cell Signaling, Beverly, MA) and then stripped and reprobed with an
`a-tubulin antibody (Neomarkers) to control for equal loading.
`Statistical analyses. Results are presented as mean F one SE. Between-
`group differences used one-way ANOVA or two-way ANOVA employing
`Holm-Sidak tests for post hoc comparisons (either pairwise or versus
`controls). In some cases, the data were normalized by taking log10 before
`statistical analyses. For all tests, the level of significance was set at P < 0.05.
`Statistical calculations were done using SigmaStat 3.1 (Jandel Scientific,
`San Rafael, CA).
`
`Results
`FTY720 inhibits S1P-driven angiogenesis. Because S1P is a
`proangiogenic factor, we tested the effects of the S1PR modulator
`FTY720 (and analogues)
`in an S1P-driven angiogenesis agar
`chamber model (see Fig. 1A and C). We carried out the experiment
`in the same manner as the VEGF-driven chamber model previously
`used to characterize the VEGFR inhibitor PTK787/ZK222584 (PTK/
`ZK; ref. 13) but substituting S1P for VEGF. The effects of FTY720
`at 3 and 0.3 mg/kg, an analogue NVP-AAL151 and its inactive
`enantiomer NVP-AAL149 both at 2.5 mg/kg, and a VEGF receptor
`tyrosine kinase inhibitor NVP-AAL993 at 100 mg/kg were assessed.
`The mice received a single administration of vehicle or compound
`4 to 6 hours before implantation and were subsequently treated
`once daily for 3 days before explanting the chamber. In this time, a
`new blood vessel–rich tissue is formed around the implanted
`chamber. This tissue was removed, weighed, and analyzed for total
`amount of hemoglobin (a measure for vascularity and hemorrhage)
`
`FTY720 Inhibits Angiogenesis
`
`and Tie-2 protein (indicative of endothelial cell amount and
`therefore vascularity only) in the tissue.
`S1P was overall a weaker promoter of the indices of angiogenesis
`in this chamber model compared with VEGF (Fig. 1). FTY720 at
`doses of 0.3 and 3 mg/kg reduced the weight of newly formed tissue
`and its hemoglobin and blood vessel content in the S1P-driven
`agar implant model (Fig. 1A). NVP-AAL151 also functioned as an
`inhibitor in this model, whereas NVP-AAL149 was considerably
`weaker. As expected, the VEGFR tyrosine kinase inhibitor NVP-
`AAL993 did not exert any influence on the S1P-driven angiogenesis
`model (Fig. 1C). These results show that FTY720 and NVP-AAL151
`are able to inhibit S1P-driven angiogenesis in vivo.
`FTY720 inhibits VEGF-driven angiogenesis. We further
`tested if FTY720 and its analogues at the same doses as above
`were able to inhibit VEGF-driven angiogenesis using the s.c.
`implant model
`(Fig. 1B and C). VEGF-filled agar chambers
`(Fig. 1B, gray columns) produced increased weight as well as
`amount of hemoglobin and Tie-2 in the newly formed tissue
`compared with an agar implant with no growth factor (Fig. 1B,
`black columns). FTY720 at 3 and 0.3 mg/kg inhibited the VEGF-
`dependent increased weight (P < 0.001 versus VEGF control)
`and Tie-2 (P < 0.001 versus VEGF control) and hemoglobin (P <
`0.05 versus VEGF control) content of the tissue. There was a
`tendency for the 3 mg/kg dose to be more active than the
`0.3 mg/kg dose in all of the variables evaluated. However, this
`only reached statistical significance with hemoglobin content.
`The immunosuppressive properties of FTY720 were evident by
`the reduced number of WBC, specifically lymphocytes, circulating
`in the blood (data not shown). Platelets, granulocytes, and RBC
`were unaffected (data not shown).
`With the exception of NVP-AAL149 (the inactive enantiomer
`that is not phosphorylated by sphingosine kinase; see refs. 9, 10),
`all compounds were able to inhibit the increase in weight of the
`newly formed tissue, hemoglobin, and Tie-2 content in this model
`(Fig. 1C). NVP-AAL149 was significantly less active than either
`NVP-AAL151 or NVP-AAL993 in terms of impairing tissue accu-
`mulation and Tie-2 levels (P < 0.001), hemoglobin content of
`the tissue (versus NVP-AAL151, P = 0.013; versus NVP-AAL993,
`P = 0.005). Interestingly, in this VEGF-driven angiogenesis model,
`the VEGFR inhibitor NVP-AAL993 and the S1PR signaling modu-
`lator NVP-AAL151 were not statistically different in their inhibitory
`effects (mg tissue, P = 0.9; hemoglobin levels, P = 0.3; Tie-2 levels,
`P = 0.5). These results show that at doses producing leukopenia,
`FTY720 and its analogues have potent antiangiogenic activity in a
`well-established in vivo angiogenesis model driven by two distinct
`proangiogenic factors.
`We also tested the ability of FTY720 to block neoangiogenesis in
`the mouse corneal pocket assay, a widely used angiogenesis model.
`In this model (12), VEGF pellets were surgically implanted into
`avascular corneas of C57BL/6 mice to induce a robust angiogenesis
`response (Fig. 1D). Twenty-four hours after implantation, mice were
`treated with either vehicle alone or FTY720 at 0.3 or 3 mg/kg orally
`once per day. The eyes were examined on postoperative days 3
`through 6, and the induced vascular response was measured. Oral
`treatment with FTY720 caused a marked (versus controls: 0.3 mg/kg
`FTY720, P = 0.002; 3 mg/kg FTY720, P < 0.001) and apparently dose-
`dependent inhibition of new blood vessel formation (3 mg/kg
`FTY720 superior to 0.3 mg/kg, P = 0.02).
`FTY720 reduces leakiness of blood vessels. Because blockade
`of the hemoglobin response in the chamber model may reflect an
`effect on vascular permeability rather than an effect on new vessel
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`Figure 1. Antiangiogenic effect of FTY720. A, effect of FTY720 (0.3 or 3 mg/kg orally) or vehicle (PEG300, 100% 5 mL/kg orally) on an S1P-driven chamber model
`(5 Amol/L). Weight (left), total hemoglobin (middle ), and Tie-2 levels (right) of the tissue are plotted. From two independent experiments were pooled (10-12 mice).
`Columns, mean; bars, SE. The statistical significance of inhibition compared with vehicle-treated S1P containing chambers was determined using one-way
`ANOVA with post hoc Holm-Sidak tests. B, effect of FTY720 (0.3 or 3 mg/kg orally) or vehicle (PEG300, 100% 5 mL/kg orally) on the VEGF-driven chamber
`model (2 Ag/mL). Weight (left ), total hemoglobin (middle), and Tie-2 levels (right ) of the tissue are plotted. From two independent experiments were pooled
`(10-12 mice). Columns, mean; bars, SE. Statistical significance of inhibition was determined using one-way ANOVA with post hoc Holm-Sidak tests. C, treatment
`of mice with NVP-AAL993 (100 mg/kg orally), NVP-AAL149 (2.5 mg/kg i.v.), NVP-AAL151 (2.5 mg/kg i.v.), or vehicle (PEG300, 100% 5 mL/kg orally) in an S1P-driven
`(5 Amol/L) or VEGF-driven (2 Ag/mL) agar chamber model (see top ). The animals were sacrificed for measurement of the vascularized chamber-adherent tissues
`(weight and hemoglobin and Tie-2 content) 24 hours after the last dose (n = 10-12 per group, pooled data from two independent experiments per dose).
`GF, growth factor. Statistical significance of inhibition was determined using one-way ANOVA with post hoc Holm-Sidak tests. D, C57/BL6J mouse corneal pocket
`assay. Photomicrographs showing vascularization induced by VEGF-implanted pellets after 6 days of treatment with vehicle (5% glucose in water), 0.3 mg/kg FTY720,
`or 3 mg/kg FTY720. Numbers, % inhibition of blood vessel growth (mean F SE). Both the high-dose FTY720 group (P < 0.001) and the low-dose group
`(P = 0.002) were statistically significantly different from controls (one-way ANOVA with post hoc Holm-Sidak test). The experiment was done two separate times.
`Representative experiment. Quantitative data pooled from both experiments (n = 6 per group).
`
`formation, we investigated the effect of FTY720 in a specific assay
`for vascular permeability (Fig. 2). For this purpose, VEGF-soaked
`beads were implanted s.c. into one ear of a mouse, which produces
`new but leaky and torturous vessel formation over a period of
`2 days. The other ear of the same mouse is implanted with PBS
`soaked beads as a control. After 2 days, Evans blue is injected i.v.,
`and images of the ears are taken. Blood vessel leakiness is assessed
`initially by visual
`inspection (Fig. 2A) and by image analysis
`determination of the area of extravasated Evans blue dye (Fig. 2A
`and B). Vehicle, PTK/ZK (100 mg/kg), or 0.3 or 3 mg/kg FTY720
`were administered 2 hours before Evans blue injection. Two-way
`ANOVA indicated that VEGF increased vessel leakiness compared
`
`with that produced by control beads (P < 0.001), and both
`compounds alone inhibited both basal and VEGF-induced vascular
`permeability changes. At both 0.3 and 3 mg/kg, FTY720 inhibited
`basal leakiness (likely to be caused by wounding during implan-
`tation or inflammatory processes associated with the intracorporal
`presence of a foreign body; Fig. 2A and B) as well as VEGF-induced
`leakiness of the vessels. As expected, the two VEGF receptor
`tyrosine kinase inhibitors NVP-AAL993 (not shown) and PTK/ZK
`also effectively reduced leakiness of the vasculature in this model
`(Fig. 2). When comparing (two-way ANOVA) the effect of treat-
`ment in the absence of VEGF stimulus, PTK/ZK or FTY720 at
`0.3 or 3 mg/kg inhibited vascular leakiness, but these treatments
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`were not significantly different from each other (all Ps < 0.05). The
`presence of VEGF clearly promoted vascular permeability. Within
`the VEGF-treated groups, PTK/ZK and 0.3 mg/kg FTY720 impaired
`vascular permeability by (P = 0.022 and P < 0.001 versus controls),
`and 3 mg/kg FTY720 produced an effect greater than either of
`the other two active treatments (Fig. 2B). These results indicate
`that part of the observed pharmacologic antiangiogenic activity of
`FTY720 may be due to abrogation of the increases in vascular
`permeability caused by VEGF.
`FTY720 pretreatment inhibits S1P-stimulated migration
`and calcium mobilization in vitro. Because FTY720 was recently
`shown to act as a functional antagonist on S1P1 receptors on
`T cells, inhibiting their egress from lymph nodes (16), we assessed
`whether FTY720 can also modulate the response of endothelial
`cells to S1P. We first examined the effect of FTY720, FTY-P, NVP-
`AAL151, and NVP-AAL149 on S1P-driven endothelial migration
`in vitro (data not shown). Compounds were preincubated with
`HUVEC for 30 minutes before the cells were allowed to migrate
`towards S1P (500 nmol/L) in the continuing presence of com-
`pounds. FTY720, FTY-P, and NVP-AAL151 were all able to
`significantly inhibit S1P-induced HUVEC migration with IC50
`values of 6.5, 2.5, and 7.4 nmol/L, respectively, whereas NVP-
`AAL149 was inactive at all doses tested (>250 nmol/L). Because
`the prodrug FTY720 was active in this assay, this suggested that the
`4-hour incubation time of the assay was sufficient for conversion
`of FTY720 to FTY-P by sphingosine kinase and for the latter form
`to subsequently inhibit the S1P-mediated migration of cells (17).
`Activation of S1PR by S1P causes G-protein–coupled activation
`and mobilization of calcium from the endoplasmic reticulum (9, 18).
`
`We tested FTY720 and its analogues in this assay to see if they could
`antagonize S1P-induced Ca2+ mobilization (data not shown). The
`compounds were preincubated with HUVEC for 3 hours before the
`assay to allow the prodrugs to be phosphorylated by sphingosine
`kinase. FTY-P, FTY720, and NVP-AAL151 were all able to block the
`S1P-driven calcium mobilization with IC50 values of 55, 164, and 156
`nmol/L, respectively, whereas NVP-AAL149 was unable to inhibit
`the S1P response (>34.4 Amol/L). The IC50 differences observed
`between the migration assay and Ca2+ mobilization are probably
`due to the longer incubation period (FTY720 converting to FTY-P)
`in the migration assay as well as the difficulty of inhibiting the
`amplification of signaling in the Ca2+ mobilization assay.
`FTY-P internalizes S1P1. To investigate the mechanism by
`which FTY720 and its analogues are modulating the S1P-driven
`responses, we incubated HEK293 cells stably expressing an S1P1-
`GFP fusion protein (15) with 10 nmol/L of either S1P or FTY-P for
`60 minutes and analyzed the localization of S1P1 by confocal
`fluorescence imaging. S1P1-GFP is normally expressed on the cell
`surface (Fig. 3A). Addition of S1P at 10 nmol/L did not affect
`the localization of this receptor; however, addition of FTY-P at the
`same concentration resulted in the internalization of S1P1 as
`detected by the punctated endosomal appearance for S1P1. An
`additional experiment was designed to investigate recycling of the
`receptor back to the surface of HEK293 cells (Fig. 3B). In this case,
`100 nmol/L of S1P or FTY-P were added to the cells. This high dose
`of ligand resulted in receptor internalization in both cases after
`60 minutes, although a significantly greater internalization was
`observed with the FTY-P treatment. The ligands were subsequently
`washed out, and the cells were allowed to recover for 60 minutes.
`
`Figure 2. FTY720 reduces leakiness of
`vessels. A, photomicrographs showing leakage
`of Evans blue from vessels with implanted
`PBS-soaked beads (top ) and VEGF-soaked
`beads (bottom ). Treatment with FTY720,
`PTK/ZK, or vehicle. Bar, 1 mm. This
`experiment was done twice. Representative
`experiment. B, measurements of the dye
`extravasation area (mm2) were carried out
`using computer-assisted image analysis
`software. Columns, mean; bars, SE. P s are
`from one-way ANOVA with post hoc
`Holm-Sidak tests.
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`Figure 3. FTY-P internalizes S1P1, does not affect VEGFR levels at the endothelial cell surface, and does not affect VEGF induced pMAPK. A, HEK293 cells
`transfected with S1P1-GFP were incubated with 10 nmol/L of either S1P or FTY-P, or vehicle (C ) for 60 minutes before imaging for S1P1-GFP in a confocal microscope.
`B, HEK293 cells transfected with S1P1-GFP were cultured together with 100 nmol/L of either S1P or FTY-P for 1 hour. Some representative wells were imaged.
`The remaining wells were washed briefly and incubated with medium minus the ligands for a further hour before imaging. C, FTY-P treatment does not lead to VEGFR2
`internalization. HUVECs were analyzed for cell surface VEGFR2 levels by FACS analysis following 30 minutes of incubation with FTY-P or vehicle. Control
`(omission of primary antibody) and FTY-P at 0, 10, and 100 nmol/L. Positive cell surface staining for VEGFR2 (gray area). Effect of incubation for 30 minutes with
`FTY-P at 10 nmol/L (red ) and 100 nmol/L (blue ) on VEGFR2 surface level. Black line, antibody control. D, pMAPK Western blot analysis in HUVECs; 500 nmol/L
`of FTY720 (FTY ), FTY-P, or PTK/ZK were incubated for 20 minutes