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Complete and Specific Inhibition of Adult LymphaticRegeneration by a Novel VEGFR-3 Neutralizing Antibody

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Angiogenesis (bloodvessel growth), lymphangiogenesis (lymph system growth) are allintrinsicallyconnected with lymphedemaand share many of the same genes. Wehave several pages onboth processes.

PatO'Connor

May23, 2008

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Complete and Specific Inhibition of Adult LymphaticRegeneration by a Novel VEGFR-3 Neutralizing Antibody

Journalof the National Cancer Institute, Vol. 97, No. 1, 14-21,January 5, 2005
DOI: 10.1093/jnci/dji003

BronislawPytowski, Jeremy Goldman, KrisPersaud, Yan Wu, LarryWitte, Daniel J.Hicklin, Mihaela Skobe, KendrickC. Boardman, MelodyA. Swartz

Affiliationsof authors: Molecular and CellularBiology, ImClone Systems, New York, NY (BP, KP, LW); BiomedicalEngineeringDepartment, Northwestern University, Evanston, IL (JG, KCB, MAS);ExperimentalTherapeutics, ImClone Systems, New York, NY (YW, DJH); Derald H.RuttenbergCancer Center, Mt. Sinai School of Medicine, New York, NY (MS)

Correspondenceto: Melody A. Swartz, PhD, AssistantProfessor, Institute for Biological Engineering and Biotechnology,School ofLife Sciences/LMBM/AAB041, Swiss Federal Institute of TechnologyLausanne (EPFL),1015 Lausanne, Switzerland (e-mail: melody.swartz@epfl.ch)

 ABSTRACT
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 
Background: New lymphatic growth may contribute totumor metastasis.Activation of vascular endothelial growth factor receptor 3(VEGFR-3)by its ligands VEGF-C and -D is necessary for embryonic andtumorlymphangiogenesis. However, the exact role of VEGFR-3 signalinginadult lymphangiogenesis and in lymphatic vessel survivalandregeneration is unclear. Methods: A novel ratmonoclonal antibodyto murine VEGFR-3, mF4-31C1, which potently antagonizes thebindingof VEGF-C to VEGFR-3, was developed. We tested the effectsofsystemic mF4-31C1 administration in a mouse tail skinmodel oflymphatic regeneration, either with or without localoverexpressionof VEGF-C, and we observed lymphatic and bloodvessel regenerationover time using microlymphangiography andimmunostaining. Results:Normal mice regenerated complete and functionallymphatic vesselswithin 60 days of surgery. In athymic miceimplanted withVEGF-C-overexpressing human breast carcinomacells, lymphaticregeneration took place over 25 days andresulted in hyperplasticvessels. Under either condition, no lymphaticregeneration occurredin mice receiving mF4-31C1 during theregeneration period. Bloodangiogenesis and preexisting lymphatic vesselswere unaffected, bothin morphology and in function. Conclusions:Blocking VEGFR-3completely and specifically prevented bothphysiologically normal andtumor VEGF-C-enhanced lymphangiogenesis in theadult mouse but had noeffect on either blood angiogenesis or thesurvival or function ofexisting lymphatic vessels. Thus, targetingVEGFR-3 with specificinhibitors may block new lymphatic growthexclusively.
  INTRODUCTION
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 
The lymphatic system provides a pathway for metastatic tumorcellmigration and growth, and many recent studies have provided insightinto the molecular regulation of lymphangiogenesis in tissuedevelopment as well as tumor growth and invasion (13).Vascular endothelial growth factors C and D (VEGF-C and -D)bindto and activate VEGF receptor 3 (VEGFR-3) by triggering itsphosphorylation (4,5) and also bindto VEGFR-2 (in only its fully processed form (6)),whose primary ligand is VEGF-A. VEGFR-3 isprimarily expressed onlymphatic endothelium (7),and increasedexpression of VEGF-C in tumors is related to an increasednumber andsize of tumor-associated lymphatic vessels aswell as increasedmetastasis (810). Consequently,it has been hypothesized that antagonists of VEGFR-3function mightinhibit tumor metastasis by preventing tumor lymphangiogenesis(1,10,11).However, it is unclear whether new lymphatic growth couldbe blockedwithout affecting normal lymphatic function becauseit has beensuggested that VEGFR-3 signaling is necessary forlymphatic survival (12).

Anumber of studies have been conducted to determine the effectsonlymphangiogenesis of blocking VEGFR-3 activation. For example,lymphaticdevelopment was delayed in the skin of transgenic miceexpressingsoluble VEGFR-3 (13)and lymphatic drainage wasreduced at the periphery of VEGF-C-overexpressing tumors towhichsoluble VEGFR-3 was delivered by adenovirus (10).Furthermore, exogenous fibroblast growth factor2 (FGF-2) and VEGF-Cwere both found to induce lymphangiogenesis inthe mouse cornea;the effects of FGF-2 were decreased, but not eliminated, bytreatmentwith AFL4, a rat monoclonal antibody (mAb) with specificity formurine VEGFR-3 (14).AFL4 was also shown to inhibitblood angiogenesis in tumors (15). Despitethese findings, it remains to be shown whetherlymphangiogenesis canbe specifically and completely blocked in aphysiologically relevantadult model without affecting either bloodangiogenesis orpreexisting lymphatic vessels.

Toelucidate the specific role of VEGFR-3 signaling in tissue regeneration,we used a novel rat mAb, mF4-31C1, in an adult modeloflymphangiogenesis. We have previously reported the productionof amAb, hF4-3C5, which antagonizes the activation ofhuman VEGFR-3 byVEGF-C (16).However, hF4-3C5 does not cross-reactwith murine VEGFR-3. We first determined whether mF4-31C1could blockthe activation of murine VEGFR-3 by VEGF-C. Next, we used mF4-31C1ina recently developed adult mouse model of skin regeneration (17,18)that uniquely enabled us to observe the process of lymphangiogenesisinterms of both physiologic function and biology and to differentiatenewlymphatic growth from preexisting lymphatic vessels. This mousemodelallowed us to alter the biochemical environment directly,in thiscase, by implanting VEGF-C-overexpressing tumorcells within thecollagen scaffold in the mouse tail prior togelation and skinregeneration. We then tested if neutralization ofVEGFR-3 signalingby systemic administration of mF4-31C1 couldprevent lymphaticregeneration in the presence of excess exogenousVEGF-C that wassecreted by implanted tumor cells.
  MATERIALS AND METHODS
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Notes
Abstract
Introduction
Materials andMethods
Results
Discussion
References
 
Construction and Expression of Human VEGF-C{Delta}N{Delta}C

cDNAencoding the fully processed region of human VEGF-C (6)(VEGF-C{Delta}N{Delta}C;spanning amino acids T at position 103 to L at position 215)wasprepared by polymerase chain reaction using cDNA from humanumbilicalvein endothelial cells. The cDNA was cloned intothe vector pSecTag2B(Invitrogen) and transfected into Chinesehamster ovary cells. VEGF-C{Delta}N{Delta}Cprotein containing C-terminal vector-derivedpolyhistidine tag waspurified using Ni2+ chromatography. RecombinantVEGF-C{Delta}N{Delta}Crecapitulates the natural product of the proteolyticcleavage ofnascent VEGF-C at the N and C termini withmaximal affinity forVEGFR-3 (6).Within the mature region, theamino acid sequences of the human and the murine VEGF-C{Delta}N{Delta}Cproteins were 94% identical.

Binding and Blocking Assays

Afusion protein consisting of the soluble extracellular domainofmurine VEGFR-2 (sR2-AP) fused to the human-secreted alkalinephosphatase(AP) protein was created using the expression vector AP-Tag(19)and purified as previously reported (16,20).The expression vector for the solubleextracellular domain of murineVEGFR-3 (sR3-AP) was made using nucleotides 26–2363 ofthemurine VEGFR-3 (accession number L07296).sR3-AP was expressed and purified as reportedfor sR2-AP (20).In vitro binding and blocking assays wereperformed as describedpreviously, except that murine sR3-AP or sR2-APwas used in place ofhuman sR3-AP (16).Recombinant human VEGF-C{Delta}N{Delta}Cwas used in all assays. The binding kinetics ofsR3-AP or the mAbmF4-31C1 were measured by surface plasmonresonance on the BIACORE2000 biosensor (BIACORE, Piscataway, NY). VEGF-C{Delta}N{Delta}Cor soluble VEGFR-3 (sR3-AP) was immobilized ona sensor chip, andeither sR3-AP or the mAb mF4-31C1 was injectedover the surface ofthe sensor at various concentrations. Sensogramswere evaluated usingthe BIA Evaluation 3.2 program to determine thebinding rateconstants.

Generation of Rat mAbs toMurine VEGFR-3

Lewisrats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were primedwith a subcutaneous injection of 100 mg of mR3-AP in completeFreund’s adjuvant (Sigma). Rats received four intraperitonealbooster injections of 100 mg of mR3-AP at 2-week intervals.Ratswhose sera showed the highest titer of inhibition inthe VEGFR-3blocking assay (see below) were injected intravenously withanadditional 50 mg of sR3-AP. After 5 days, splenocytes wereharvestedand fused with mouse myeloma cells P3-X63-Ag8.653. Hybridomasweregenerated and subcloned according to standard protocols(21).Hybridomas secreting antibodies that bound to immobilizedmR3-AP werefurther tested; anti-VEGFR-3 antibodies wereselected based onpositive binding to immobilized sR3-AP andfurther analyzed in thecompetitive VEGF-C blocking assay.

Receptor Phosphorylation

eEndcells, an immortalized line of murine endothelial cells (akind gift of Dr. Michael Pepper, University of Geneva MedicalCenter)(22) wereserum starved overnight and incubated for30 minutes in the presence or absence of mF4-31C1, nonimmuneratimmunoglobulin G (IgG), or AFL4 prior to stimulation for 15minuteswith 100 ng/mL of either the 165-amino acid isoform ofVEGF (VEGF165)(R&D Systems, Minneapolis, MN) or VEGF- C{Delta}N{Delta}C.VEGFR-3 was immunoprecipitated from cell lysates using mF4-31C1andprotein G Sepharose resin (Amersham Biosciences, Uppsala,Sweden).Immunoprecipitated proteins were resolved by4%–20% sodium dodecylsulfate (SDS)—polyacrylamide gelelectrophoresis andelectrophoretically transferred to nitrocellulose membranes.Phosphotyrosine residues were detected by immunoblotting withthe mAbPY-20 (Transduction Laboratories, Lexington, KY). TotalVEGFR-3 wasdetected with a rabbit polyclonal antibody tomouse VEGFR-3 (M-20,Santa Cruz Biotechnology, Inc., Santa Cruz,CA). mAb AFL4 waspurchased from eBiosciences (San Diego, CA).

Mitogenic Assays with Cells Expressing ChimericVEGFR-3-cFMS Receptor

cDNAencoding the extracellular domain of mouse VEGFR-3 was fusedwith cDNA encoding the transmembrane and cytoplasmic domainsof thereceptor for human colony-stimulating factor 1 (cFMS) intheexpression vector pIres (Invitrogen). The DNA was electroporatedintoNIH-3T3 mouse fibroblast cells, and cell clones were selectedbygrowth in G418. Plasma membrane expression of VEGFR-3-cFMS wasdemonstrated using indirect immunofluorescence with antibodiesspecificfor murine VEGFR-3. Mitogenic assays were performed asdescribedpreviously (16).Cells (5 x103 per well) were platedonto 96-well tissue cultureplates (Wallach, Inc., Gaithersburg, MD) andincubated in serum-freemedium at 37°C for 72 hours. Various amounts ofantibodies wereadded and preincubated at 37°C for 1 hour,after which VEGF-C{Delta}N{Delta}Cor VEGF165 was added toa final concentration of 20 ng/mL.After 18 hours of incubation, 0.25 mCi of [3H]thymidine(Amersham)was added to each well and incubated for anadditional 4 hours. Thecells were placed on ice, washed once withserum-containing medium,incubated 10 minutes at 4°C with 10%tricholoroacetic acid, andsolubilized in 25 µL of 2% sodiumdodecylsulfate. Incorporatedradioactivity was measured with a scintillationcounter (Model 1450Microbeta Scintillation Counter, Wallach).

Lymphangiogenesis Model

Werecently developed a model of lymphangiogenesis in regeneratingthetail skin of adult mice (18).For all studies,6-8-week-old female Balb/c and athymic mice(Charles River Labs,Wilmington, MA) were used; three to five micewere used for eachcondition at each time point examined. Micewere anesthetized with asubcutaneous injection of ketamine (100 mg/kg)and xylazine (10mg/kg). Postsurgical analgesic (buprenorphine,2 mg/mL) wasadministered twice daily for 1 week bysubcutaneous injection. Allprotocols were approved by the Animal Care andUse Committee ofNorthwestern University.

Theregenerating region of skin was created as previously described(18).Briefly, a 2-mm-wide circumferential band of dermal tissue (inwhichthe lymphatic network in the tail skin is contained) wasexcisedmidway up the tail, leaving the underlying bone, muscle,major bloodvessels, and tendons intact. The area was thencovered with aclose-fitting, gas-permeable silicone sleeve andfilled with type Irat tail collagen. The collagen provided acontrolled environment inwhich skin could regenerate, and any lymphaticendothelial cells orstructures later observed within this regionwere the result of newlyinitiated cell migration, proliferation, andorganization.

Twovariants of the model were used: 1) normal physiologic lymphaticregenerationin adult Balb/c mice and 2) lymphatic regeneration inthe presence ofexcess tumor-derived VEGF-C in athymic mice. Inthe latter,VEGF-C-overexpressing or control-transfected humanbreast carcinomacells (MDA-MB-435) (9)were implanted at 1 x106 cells/mL within the collagen scaffold.Lymphatic regenerationwas ascertained in Balb/c mice at 60 days and in tumor-bearingathymic mice at 25 days postsurgery (see below). Ingroups receivingmF4-31C1, the antibody was administered at 25µg/g every 2 days byintraperitoneal injection beginning the day ofsurgery and proceedinguntil termination of the experiment.

Detection of Functional Lymphatic Vessels ViaMicrolymphangiography

Tovisualize lymph flow patterns both in situ as well as postfixationinthin sections, the animal was anesthetized and a 1% solutionoftetramethylrhodamine isothiocyanate (TRITC)-conjugated, lysine-fixabledextranof 2 x 106 Da (Molecular Probes,Eugene, OR) was injected intradermally into thetail tip where it wastaken up and transported by the lymphaticvessels in the proximaldirection (23),revealing fluid channelsand functional lymphatic vessels. The anesthetized animalwas thenkilled with a perfusion through the blood vasculature withZamboni’s fixative via the abdominal aorta (18).The tail was snap frozen in liquid nitrogen, stored at–80°C, andlater cryosectioned. This fixation procedure resulted in crosslinkingthe dextran fluid tracer in place, thereby allowing lymphfluid to bevisualized in cryosections and correlated tofunctional lymphaticvessels (e.g., dextran tracer colocalized withlymphatic endothelialcells).

Immunofluorescence and Immunohistochemistry

Tailspecimens were cut into 10-µm longitudinal cryosections andimmunostained. To detect lymphatic endothelial cells, a rabbitpolyclonal antibody against the lymphatic-specific hyaluronanreceptorLYVE-1 (24)(kind gift from Dr. David Jackson, JohnRadcliffe Hospital, Oxford, U.K.) was used along with abiotinylated goatanti-rabbit secondary antibody (Dako) and Alexa fluor 488-conjugatedstreptavidin(Molecular Probes). To detect blood endothelial cells,fluoresceinisothiocyanate-conjugated anti-mouse monoclonal CD31antibody (PharMingen)was used. Although lymphatic endothelial cellsalso express CD31, theexpression is very weak compared with that ofblood endothelial cells(18) and thetwo cell types could bereadily differentiated based on staining intensity. Asa negativecontrol, normal skin was costained for CD31 and LYVE-1;no visiblecolocalization occurred (data not shown). Cellnuclei were labeledwith 4',6-diamino-2-phenylindole (DAPI) (VectorLabs). To detect VEGF-Cprotein, sections were incubated with a goatanti-mouse antibodyagainst VEGF-C (Santa Cruz Biotechnology) witha biotinylated rabbitanti-goat secondary antibody and Vector Redsubstrate (Vector Labs);nuclei were counterstained with hematoxylin.

Statistical Methods

Atleast three sections were counted per mouse with three to fivemice per group. Data were presented as means with 95% confidenceintervals(CIs). All P values were calculated using atwo-sided Student’st test.
  RESULTS
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 
mAb mF4-31C1 Characteristics

Webegan by generating a mAb to murine VEGFR-3. Hybridoma clonesthatproduced antibodies with VEGFR-3 inhibitory activity were generatedby fusing murine myeloma cells with splenocytes from arat immunizedwith sR3-AP, a chimeric protein consisting of thesolubleextracellular domain of murine VEGFR-3 fused to thehuman-secretedAP. Conditioned media from four clones inhibited 100%,90%, 80%, and50% of the binding of sR3-AP to immobilized VEGF-C.Stable monoclonalhybridoma cell lines mF4-31C1 and mF4-12A10were established from thetwo clones with the highest blocking activityafter subcloning threetimes. mF4-31C1 showed consistently more potentantagonist activitythan mAb mF4-12A10 (data not shown) and waschosen for high-levelproduction and purification. The isotype ofmF4-31C1 was determinedto be rat IgG 2a. The affinity constant ofsR3-AP for the fullyprocessed VEGF-C{Delta}N{Delta}Cwas measured by BIACORE at 1 nM compared with 150 pM forthe bindingof mF4-31C1 to immobilized sR3-AP (Table 1).

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Table1. Binding kinetics of sR3-AP and mF4-31C1*

 

 
The potency of mF4-31C1 was compared using in vitro binding andblocking assays to that of AFL4, an isotype-matched rat mAbthat hasbeen reported to act as an antagonist of murine VEGFR-3(15).Both antibodies bound similarly to sR3-AP (Fig. 1A).However, mF4-31C1 potently inhibited the binding of sR3-AP toimmobilized VEGF-C{Delta}N{Delta}C;neither the AFL4 antibody nor mAb DC101, anantagonist of murineVEGFR-2 (25),had an inhibitory effect (Fig.1B). VEGF-C{Delta}N{Delta}Cis a recombinant equivalent of proteolytically fullyprocessednascent VEGF-C and has been reported to bind toand activate VEGFR-2in addition to VEGFR-3 (6).Thus, we investigatedwhether mF4-31C1 would antagonize ligand binding tomurine VEGFR-2.The addition of mF4-31C1 had no effect on theinteraction of eitherVEGF-C{Delta}N{Delta}Cor the most abundant form of VEGF-A, VEGF165,with solublesR2-AP (Fig. 1, C and D),demonstrating thespecificity of this antibody for the murine VEGFR-3. In contrast,mAbDC101 inhibited the binding of both VEGF-C{Delta}N{Delta}Cand VEGF165 to VEGFR-2with nearly identical potency.

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Fig.1. In vitro characterization of the rat monoclonal mF4-31C1antibody binding to vascular endothelial growth factor (VEGFR-3). A)Various amounts of mF4-31C1 (open circles) or therat anti-VEGFR-3 AFL4 antibody (filled triangles)were added to 96-well plates coated with soluble VEGF-3–alkalinephosphatase (sR3-AP). Bound antibodies were detected with aperoxidase-labeled rabbit anti-rat IgG antibody. B)Saturating amounts of sR3-AP were mixed with various amounts of theantibodies and added to 96-well plates coated with fully processedVEGF-C (VEGF- C{Delta}N{Delta}C). The amount ofbound receptor was measured as shown in panel A. mF4-31C1 (opencircles, 50% inhibitory concentration = 1 nM), AFL4 (filledtriangles), and the anti-murine VEGFR-2 antagonist mAb DC101 (filledcircles). Saturating amounts of soluble VEGFR-2–AP (sR2-AP)were mixed with various amounts of antibodies and added to 96-wellplates coated with C) the fully processed region ofVEGF-C (VEGF-C{Delta}N{Delta}C) or D)the 165 amino acid isoform of VEGF (VEGF165).The amount of bound receptor was measured as in panel A. DC101 (filledcircles) and mF4-31C1 (open circles).Graphs depict means and 95% confidence intervals from two independentdata sets.

 

 
Effect of mF4-31C1 on VEGF-C-Stimulated Phosphorylationof VEGFR-3

Weutilized the immortalized murine endothelial cell line eEnd (22)to determine the capacity of mF4-31C1 to antagonize the VEGF-C-stimulatedactivation of VEGFR-3. Stimulation of serum-starved eEndcells withrecombinant human VEGF-C{Delta}N{Delta}Cresulted in the strong phosphorylation ofVEGFR-3 (Fig. 2).In contrast, the addition of VEGF165had no effect onVEGFR-3 phosphorylation. VEGF-C{Delta}N{Delta}C-mediated VEGFR-3 phosphorylation was blocked in adose-dependent mannerby mF4-31C1; mAb AFL4 had no effect (Fig. 2).The additionof mF4-31C1 to unstimulated cells did not phosphorylateVEGFR-3, evenat the highest dose used (Fig. 2).


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Fig.2. Inhibition of vascular endothelial growth factor-C(VEGF-C)-stimulated VEGFR-3 phosphorylation by mF4-31C1. Serum-starvedimmortalized murine endothelial (eEnd ) cells were stimulated by theaddition of 100 ng/mL of the 165 amino acid isoform of VEGF (VEGF165, A, lane 1) or fully processed region of VEGF-C (VEGF- C{Delta}N{Delta}C,C, lanes 2–6 and 8). mAbs mF4-31C1 (lanes 3–7) and AFL4 (lane 8) wereadded at the indicated concentrations 30 minutes prior to the additionof the ligands. Endogenous VEGFR-3 was immunoprecipitated from celllysates using mF4-31C1 and protein G Sepharose resin.Immunoprecipitated proteins were resolved by 4%–20% SDS—polyacrylamidegel electrophoresis and electrophoretically transferred tonitrocellulose membranes. Phosphotyrosine residues were detected byimmunoblotting with the mAb PY-20 (top). Equalloading of the wells was demonstrated by reprobing the blot with arabbit polyclonal antibody to murine VEGFR-3, M-20 (bottom).p175 and p198 = unprocessed glycosidation variants of VEGFR-3. p125 =fragment of proteolytically processed VEGFR-3 containing thetransmembrane and cytoplasmic domains.

 

 
Effect of mAb mF4-31C1 on the VEGF-C{Delta}N{Delta}C-Stimulated Mitogenic Response

Theability of mF4-31C1 to inhibit VEGF-C{Delta}N{Delta}C-stimulated signal transduction was testedusing an NIH-3T3 cell linethat expresses VEGFR-3 fused to thetransmembrane and cytoplasmicdomains of the human receptor forcolony-stimulating factor 1 (cFMS).Fluorescence-activated cell sorting analysisshowed that the chimericreceptor was localized on the plasma membraneof transfected but notparental cells (data not shown). Theincorporation of [3H]thymidineby the NIH-3T3 cells expressing VEGFR-3-cFMSwas stimulated fourfoldby the addition of VEGF-C{Delta}N{Delta}Cbut not VEGF165 (Fig. 3A).The mitogenic responsewas specifically blocked in a dose-dependent manner bymAb mF4-31C1with a 50% inhibitory concentration of 1 nM (Fig.3B).In contrast, mAb AFL4 did not inhibit the mitogenic responseof cellsexpressing VEGFR-3-cFMS to VEGF-C{Delta}N{Delta}Cat any concentration used.


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Fig.3. Inhibition of vascular endothelial growth factor-C(VEGF-C)-mediated mitogenic response by mAb mF4-31C1. A)Mitogenic response of NIH-3T3 mouse fibroblast cells that expresschimeric murine VEGFR-3–human colony-stimulating factor 1 (VEGFR-3-FMS)receptors after treatment with the fully processed region of VEGF-C(VEGF-C{Delta}N{Delta}C, filledtriangles) or with the 165 amino acid isoform of VEGF (VEGF165, open diamonds).B) Cellsexpressing murine VEGFR-3-FMS were stimulated with 80 ng/mL of VEGF-C{Delta}N{Delta}Cin the presence of various amounts of mAb mF4-31C1 (opencircles) or AFL4 (filled triangles). mAbmF4-31C1 inhibited [3H]thymidine uptake by thecells (50% inhibition at 1 nM). Means and 95% confidence intervals fromtwo independent data sets are shown. Error bars represent 95%confidence intervals.

 

 
Effects of mF4-31C1 on Normal and VEGF-C-EnhancedLymphatic Regeneration

Havingestablished mAb mF4-31C1 as a potent and unique antagonist ofmurine VEGFR-3 activation, we used this antibody to investigatetheeffects of VEGFR-3 inhibition on physiologically normal lymphangiogenesis.To this end, we used the mouse tail skin modelof lymphangiogenesisin regenerating skin (18).Sixty days aftercollagen implantation, the lymphatic vessels of untreated micehadconsistently and completely regenerated with nearly normalcapillaryarchitecture. Microlymphangiography showed thatlymphatic transportwas restored through the regenerated region,with continuity from thedistal to proximal lymphatic capillary network(Fig. 4A).In a corresponding cryosection immunostainedfor LYVE-1, the locationof lymphatic endothelial cells in theregenerating region overlappedwith the location of lymph fluid tracer (i.e.,TRITC-dextran),confirming that the lymphatic vessels in thisregion were functional(Fig. 4B). Incontrast, in mice receiving mF4-31C1by intraperitoneal injection over a 60-dayperiod, lymphaticcontinuity was not restored (Fig. 4C) and lymphaticendothelial cells were rarely found within theregenerating region(Fig. 4D), although preexistinglymphatic vesselscontinued to function normally and none of themice developed edema.Furthermore, the TRITC-dextran lymph tracer inthe regeneratingregion did not overlap with lymphatic structures,indicating thatlymph fluid was being transported mainly byinterstitial convectionthrough the region. Although the distal(upstream) and proximal(downstream) native lymphatic capillaries weredisconnected by theregenerating region, the proximal vessels couldbe seen draining thelymph from the distal region, demonstrating thefunctional competencyof both distal and proximal mature lymphaticvessels (Fig. 4,A and C).


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Fig.4. Lymphatic function as demonstrated withmicrolymphangiography and lymphatic endothelial cell staining in 10-µmcryosections by lymphatic-specific hyaluronan receptor (LYVE-1)immunofluorescence at 60 days postsurgery (see methods) in the tails ofBalb/c mice. A) Microlymphangiography usinginjection with tetramethylrhodamine isothiocyanate (TRITC)–conjugated,lysine-fixable dextran (red) and subsequent fixationand B) LYVE-1 staining (green)with the same lymph fluid tracer (red) in the tailskin of untreated mice demonstrate the functional connection todownstream lymphatic vessels and the colocalization of lymph fluid andlymphatic structures (indicated by arrows) in theregenerating region. The typical hexagonal lymphatic architecture hasreestablished in the distal part of the regenerating region. C)Microlymphangiography using TRITC-dextran (red) and D)LYVE-1 staining (green) with the same lymph fluidtracer (red) in tail skin in mF4-31C1-treated micereveal that in the regenerating region, lymph tracer is notfunctionally connected to downstream lymphatic vessels, lymphaticcapillaries have not reestablished, and almost no lymphatic endothelialcells are present. Lymph flow is shown left to right, and dashedwhite lines indicate location of the regenerating region.Cell nuclei were counterstained with 4', 6 –diamino-2-phenylindole (blue).Bar in panel C = 1 mm; bar in panel D = 500 µm.

 

 
Immunohistochemical analysis verified that implantedVEGF-C-overexpressing tumorcells within the collagen matrix substantially elevated thelevel ofVEGF-C in the regenerating region throughout the 25-dayperiod (Fig. 5B,compared with Fig. 5A, which waswithout implantedcells). In contrast to Balb/c mice, in which lymphatic regenerationrequired 60 days, lymphatic function was reestablished within25 daysin control athymic mice implanted with VEGF-C-overexpressingtumorcells in the regenerating region (Fig. 6, Aand B). The fastertimescale of lymphatic regeneration in the latter group wasdueprimarily to the presence of the tumor cells, which secretenumerousgrowth factors and cytokines to speed tissue regeneration.Theexpedited regeneration was also seen with control-transfectedtumorcells (data not shown). In addition, thelymphatic vessels weredistinctly enlarged (Fig. 6B),with roughly twiceas many cell nuclei per structure as control counterparts (e.g.,athymic mice without implanted tumor cells; data not shown).Administration of mF4-31C1 for 25 days entirely prevented theregeneration of lymphatic vessels (Fig. 6,C and D), and preexistinglymphatic vessels retained their native hexagonal architectureandwere capable of both draining and transporting thefluorescence lymphtracer.

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Fig.5. Vascular endothelial growth factor C (VEGF-C) productionby tumor cells implanted into a region of regenerating skin. VEGF-Cprotein was detected by immunostaining with a polyclonal rabbitanti-VEGF-C antibody (red) at 25 days postsurgery. A)VEGF-C expression in regenerating skin of athymic mice with noimplanted tumor cells and B) VEGF-C expression (red)in skin implanted with VEGF-C-overexpressing human breast carcinomacells. Dashed white lines indicate location ofregenerating region. Distal to proximal direction is shown left toright. Bar length = 500 µm.

 

 

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Fig.6. Lymphatic function and lymphatic endothelial celldetection at 25 days postsurgery in athymic mice implanted withvascular endothelial growth factor C (VEGF-C)-overexpressing tumorcells. A) Microlymphangiography using injectionwith tetramethylrhodamine isothiocyanate (TRITC)-conjugated,lysine-fixable dextran (red) and subsequent fixationand B) lymphatic-specific hyaluronan receptor(LYVE-1) staining (green) with the same lymph fluidtracer (red) in mouse tail skin (implanted withVEGF-C-overexpressing cells but not treated with mF4-31C1) show that inthe regenerating region, fluid channels were functionally connected todownstream lymphatic vessels and often colocalized with lymphaticendothelial cells (indicated by arrowheads). Manylymphatic structures are hyperplastic (indicated by arrows).C) Microlymphangiography using TRITCdextran (red) and D) LYVE-1staining (green) with the same lymph fluid tracer (red)in mouse tail skin implanted with VEGF-C-overexpressing human breastcarcinoma cells and treated with mAb mF4-31C1 show that in theregenerating region, lymph flow was not functionally connected betweenupstream and downstream lymphatic vessels, and almost no lymphaticendothelial cells could be seen. Longitudinal cryosections were 10 µmthick, and cell nuclei were counterstained with 4', 6–diamino-2-phenylindole (blue). Lymph flow is shown left to right, anddashed white lines indicate location of the regenerating region. PanelC, bar = 1 mm; panel D, bar = 500 µm.

 

 
Morphometric analysis of immunohistologic data showed highlystatisticallysignificant differences in lymphatic endothelial celldensity betweensaline-treated and mF4-31C1-treated groups ofmice undergoing eithernormal physiologic lymphatic regeneration (meandensity = 175 and 95%CI = 125 to 225 in untreated mice, and meandensity = 10 and 95% CI =6 to 14 in treated mice; P =.003, Fig. 7A)or accelerated regeneration due to VEGF-C overexpression(meandensity = 316 and 95% CI = 271 to 361 in untreatedmice, and meandensity = 23 and 95% CI = -12 to 58 in treatedmice; P = .001,Fig. 7B).

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Fig.7. Quantification of the lymphangiogenic and angiogenicresponse to monoclonal antibody mF4-31C1 administration. Lymphatic andblood endothelial cells (LECs and BECs) were identified bylymphatic-specific hyaluronan receptor LYVE-1 and anti-mouse CD31antibody staining, respectively, together with cell nuclei(counterstained with 4', 6 –diamino-2-phenylindole). A)Normal mice were treated with saline (open bars) orthe anti-vascular endothelial growth factor receptor-3 (VEGFR-3) mAbmF4-31C1 (filled bars). Density of lymphaticendothelial cells (P = .003, treated versusuntreated, using a two-sided Student’s t test) andblood endothelial cells (P = .35, treated versusuntreated) in the regenerating region 60 days postsurgery. B)VEGF-C-overexpressing human breast carcinoma cells were implanted intoathymic mice. The number of lymphatic endothelial cells (P= .001 treated versus untreated, using a two-sided Student’s ttest) blood endothelial cells (P = .38 treatedversus untreated) in the regenerating region 25 days postsurgery aftertreatment with (filled bars) or without (openbars) mF4-31C1. Graphs depict the mean and 95% confidenceintervals from three to five mice per experiment.

 

 
Effect of mAb mF4-31C1 on Blood Angiogenesis

Todetermine blood endothelial cell density, cryosections were stainedfor CD31, an endothelial cell marker expressed prominently inbloodendothelial cells (26),and strongly staining cellswere counted. A slight reduction in blood endothelial celldensity dueto mF4-31C1 treatment was observed consistently, but the magnitudeofthis effect was small compared with the effect onlymphaticendothelial cells and failed to reach statistical significanceineither Balb/c mice (P = .35, Fig. 7A)or athymic miceimplanted with VEGF-C-overexpressing tumor cells (P= .38, Fig.7B). Thus, inhibition of VEGFR-3with the mAb mF4-31C1 didnot substantially affect vascular angiogenesis in regeneratingskin,even in the presence of excess tumor-derived VEGF-C.
  DISCUSSION
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 
In this study, we characterized the specificity and antagonistpotencyof a novel monoclonal VEGFR-3 neutralizing antibody, mF4-31C1,anddemonstrated its in vivo efficacy in a unique modelof adultlymphangiogenesis. The mF4-31C1 antibody bound specificallyto murineVEGFR-3 and blocked the phosphorylation and themitogenic activity ofVEGFR-3 in vitro. Using a mouse model of dermallymphaticregeneration, we demonstrated that thecontinuous systemicadministration of mF4-31C1 completely preventedboth normal and VEGF-C-enhancedlymphangiogenesis without substantiallyaffecting the function ofmature (preexisting) lymphatic vessels or theregeneration of bloodvessels.

Asurprising finding in the present report was that mAb AFL4 wasnot a potent antagonist of murine VEGFR-3. AFL4 has been showntoreduce lymphangiogenesis in a murine corneal pocket assayin whichmicropellets containing FGF-2 induced the production ofVEGF-Cprimarily by blood endothelial cells (14),and itwas also shown to inhibit tumor growth in several mousetumor models(15). In thelatter study, an important aspect of theantitumor effect of AFL4 was the disruption of theendothelial liningof postcapillary venules and microhemorrhage within the tumortissue,suggesting a role for VEGFR-3 in stabilizing tumor vasculature.Inview of our results on AFL4’s ability to bindbut not blockVEGFR-3, it seems reasonable to suggest thatthe reported in vivoeffects of AFL4 are mediated by a nonantagonistmechanism, such asthe steric hindrance of VEGFR-3 dimerization orantibody-inducedreduction in surface receptor expression,rather than by directinhibition of ligand binding.

Themouse tail model of skin regeneration (17,18)has unique features which allowed us toincorporateVEGF-C-overexpressing tumor cells locally andto identify whether newlymphatic and blood vessels regenerated intothe collagen scaffoldthat was initially devoid of vessels.Untreated, the excesstumor-derived VEGF-C led to grosslyhyperplastic lymphatic vessels;this result is consistent with other reports ofhyperplasticlymphatic vessels in transgenic miceoverexpressing VEGF-C in theskin (27,28), inandaround VEGF-C-overexpressing tumors (9,10,29),and following VEGF-C adenoviral expression (3032).However, we did not detect an increase in thenumber of lymphaticvessels in mice with VEGF-C overexpression(data not shown), eventhough it has been suggested that excess VEGF-Cmay also induce thegrowth of new lymphatic vessels when delivered by adenovirus(3032).Our finding in this study, that VEGFR-3 blocking completelypreventedlymphatic regeneration in the presence oftumor-derived exogenousVEGF-C, is consistent with the notion thatVEGFR-3 signaling iscritical for tumor lymphangiogenesis.

Althoughlymphatic vessels completely failed to regenerate in thepresence of mF4-31C1, we did not detect any differences inregenerating blood vessels or in preexisting lymphatic vessels(eitherin their appearance or in their ability to uptake and transportlymph), suggesting that the mF4-31C1 VEGFR-3 neutralizing antibodyspecifically blocks lymphatic regeneration. Other reports havesuggested a role for VEGFR-3 signaling in stabilizing newly formedlymphatic vessels during embryonic development (13) aswell as for lymphatic endothelial cell survival in vitro (12),implying that continuous VEGFR-3 signaling may be importantforthe survival or maintenance of existing lymphatic vessels. Ourfindings in adult tissues stand in contrast to these and suggestthatVEGFR-3 signaling is not important for the survival ofmature adultlymphatic vessels. Our findings further suggest thatthe role ofVEGFR-3 signaling may be different in lymphatic developmentand adultregeneration. This finding is consistent withour recent observationthat, although interstitial fluid flow wasnecessary for establishingnormal lymphatic capillary organization in theregenerating skin,peak VEGF-C expression was seen only during theearliest stages oflymphangiogenesis (lymphatic endothelial cellproliferation andmigration), with much less expression duringthe later stages oflymphatic capillary organization and functionalintegration (18).

Inthe present study, the initiation of both physiologically normallymphangiogenesis and tumor lymphangiogenesis was prevented bythecontinuous neutralization of VEGFR-3, starting immediately uponinitiation of skin regeneration (e.g., the implantation ofthecollagen scaffold into the mouse tail skin). In a clinical setting,however, tumor lymphangiogenesis may have already occurred bythetime the tumor is identified. In order for anti-VEGFR-3 therapyto beeffective in these patients, it must diminish existingtumor-associated lymphatic vessels. In contrast to normalfunctionallymphatic vessels, which are unaffected by VEGFR-3blocking asdemonstrated here, it is possible that tumor-associated lymphaticvessels may rely on continuous VEGF-C signaling for survivalandhyperplasticity. However, it is currently unknown whethermF4-31C1administration can reduce the density or size ofexistinghyperplastic lymphatic vessels in the tumor margin.

Insummary, our observations in a unique model of adult lymphaticregenerationin mouse skin demonstrate that 1) VEGFR-3 signaling isnecessary forthe initiation of lymphatic regeneration, 2) VEGFR-3signaling maynot be required for the proper functioning ofmature lymphaticvessels, and 3) VEGFR-3 neutralization can completelyandspecifically block adult lymphangiogenesis. These findingsraise thepossibility that human VEGFR-3 may be targeted therapeuticallywithantagonist mAbs to prevent the undesirable growthof lymphaticvessels, such as tumor-induced lymphatic hyperplasiaorlymphangiogenesis.

 
  NOTES
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 
B. Pytowski and J. Goldman contributed equally to this work.

Thefollowing authors are employees of ImClone Systems and may eitherhold stock or stock options in the company: B. Pytowski, K.Persaud,Y. Wu, L. Witte, and D. Hicklin. The results reported inthispublication may impact decisions made by ImClone Systems regardingone of its potential products.

Wethank Eva Mika, Seth O’Day, Joe Rutkowski, Xenia Jimenez, andLaura Brennan for valuable assistance and Dr. David Jackson fortheLYVE-1 antibody. We also gratefully acknowledge the supportandadvice of Dr. Peter Bohlen. We are grateful to the IllinoisDivisionof the American Cancer Society, the Lurie CancerCenter ofNorthwestern University Medical School, and theNIH/NCI Breast CancerSPORE (1 P50 CA89018-01) for their financialsupport of thisproject.
  REFERENCES
Top
Notes
Abstract
Introduction
Materialsand Methods
Results
Discussion
References
 

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Manuscriptreceived August 4, 2004; revised October 15, 2003; acceptedOctober 21, 2004.

This article has been cited by other articles inHighWire Press-hosted journals:

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