Complete and Specific Inhibition of Adult LymphaticRegeneration by a Novel VEGFR-3 Neutralizing Antibody
Angiogenesis (bloodvessel growth), lymphangiogenesis (lymph system growth) are allintrinsicallyconnected with lymphedemaand share many of the same genes. Wehave several pages onboth processes.
Complete and Specific Inhibition of Adult LymphaticRegeneration by a Novel VEGFR-3 Neutralizing AntibodyJournalof the National Cancer Institute, Vol. 97, No. 1, 14-21,January 5, 2005
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: email@example.com)
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|
cDNAencoding the fully processed region of human VEGF-C (6)(VEGF-CNC;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-CNCprotein containing C-terminal vector-derivedpolyhistidine tag waspurified using Ni2+ chromatography. RecombinantVEGF-CNCrecapitulates 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-CNCproteins 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-CNCwas 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-CNCor 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.
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- CNC.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-CNCor 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).
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.
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.
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-CNCwas measured by BIACORE at 1 nM compared with 150 pM forthe bindingof mF4-31C1 to immobilized sR3-AP (Table 1).
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-CNCresulted in the strong phosphorylation ofVEGFR-3 (Fig. 2).In contrast, the addition of VEGF165had no effect onVEGFR-3 phosphorylation. VEGF-CNC-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).
Theability of mF4-31C1 to inhibit VEGF-CNC-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-CNCbut 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-CNCat any concentration used.
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).
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.
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 (30–32).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(30–32).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.
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.
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syndrome, ptosis syndrome, plus many more. Extensive database ofinformationavailable through sister site Lymphedema People.
LymphedemaPeople New Wiki Pages
Haveyou seen our new “Wiki”pages yet? Listedbelow are just asample of the more than 140 pages now listed in our Wiki section. Weare alsoworking on hundred more. Comeandtake a stroll!
Diureticsare not for Lymphedema
LymphedemaPeople Online SupportGroups
Lymphedemaand Pain Management
ManualLymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT)
InfectionsAssociated with Lymphedema
Howto Treat a Lymphedema Wound
FungalInfections Associated withLymphedema
Extraperitonealpara-aortic lymph node dissection (EPLND)
SmallNeedle Biopsy - Fine Needle Aspiration
Home page:Lymphedema People
Page Updated: Dec. 23, 2011