Affiliations of authors: Molecular and Cellular Biology, ImClone Systems, New York, NY (BP, KP, LW); Biomedical Engineering Department, Northwestern University, Evanston, IL (JG, KCB, MAS); Experimental Therapeutics, ImClone Systems, New York, NY (YW, DJH); Derald H. Ruttenberg Cancer Center, Mt. Sinai School of Medicine, New York, NY (MS)
Correspondence to: Melody A. Swartz, PhD, Assistant Professor, Institute for Biological Engineering and Biotechnology, School of Life Sciences/LMBM/AAB041, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland (e-mail: melody.swartz{at}epfl.ch)
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ABSTRACT |
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INTRODUCTION |
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A number of studies have been conducted to determine the effects on lymphangiogenesis of blocking VEGFR-3 activation. For example, lymphatic development was delayed in the skin of transgenic mice expressing soluble VEGFR-3 (13) and lymphatic drainage was reduced at the periphery of VEGF-C-overexpressing tumors to which soluble VEGFR-3 was delivered by adenovirus (10). Furthermore, exogenous fibroblast growth factor 2 (FGF-2) and VEGF-C were both found to induce lymphangiogenesis in the mouse cornea; the effects of FGF-2 were decreased, but not eliminated, by treatment with AFL4, a rat monoclonal antibody (mAb) with specificity for murine VEGFR-3 (14). AFL4 was also shown to inhibit blood angiogenesis in tumors (15). Despite these findings, it remains to be shown whether lymphangiogenesis can be specifically and completely blocked in a physiologically relevant adult model without affecting either blood angiogenesis or preexisting lymphatic vessels.
To elucidate the specific role of VEGFR-3 signaling in tissue regeneration, we used a novel rat mAb, mF4-31C1, in an adult model of lymphangiogenesis. We have previously reported the production of a mAb, hF4-3C5, which antagonizes the activation of human VEGFR-3 by VEGF-C (16). However, hF4-3C5 does not cross-react with murine VEGFR-3. We first determined whether mF4-31C1 could block the activation of murine VEGFR-3 by VEGF-C. Next, we used mF4-31C1 in a recently developed adult mouse model of skin regeneration (17,18) that uniquely enabled us to observe the process of lymphangiogenesis in terms of both physiologic function and biology and to differentiate new lymphatic growth from preexisting lymphatic vessels. This mouse model allowed us to alter the biochemical environment directly, in this case, by implanting VEGF-C-overexpressing tumor cells within the collagen scaffold in the mouse tail prior to gelation and skin regeneration. We then tested if neutralization of VEGFR-3 signaling by systemic administration of mF4-31C1 could prevent lymphatic regeneration in the presence of excess exogenous VEGF-C that was secreted by implanted tumor cells.
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MATERIALS AND METHODS |
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cDNA encoding the fully processed region of human VEGF-C (6) (VEGF-CN
C; spanning amino acids T at position 103 to L at position 215) was prepared by polymerase chain reaction using cDNA from human umbilical vein endothelial cells. The cDNA was cloned into the vector pSecTag2B (Invitrogen) and transfected into Chinese hamster ovary cells. VEGF-C
N
C protein containing C-terminal vector-derived polyhistidine tag was purified using Ni2+ chromatography. Recombinant VEGF-C
N
C recapitulates the natural product of the proteolytic cleavage of nascent VEGF-C at the N and C termini with maximal affinity for VEGFR-3 (6). Within the mature region, the amino acid sequences of the human and the murine VEGF-C
N
C proteins were 94% identical.
Binding and Blocking Assays
A fusion protein consisting of the soluble extracellular domain of murine VEGFR-2 (sR2-AP) fused to the human-secreted alkaline phosphatase (AP) protein was created using the expression vector AP-Tag (19) and purified as previously reported (16,20). The expression vector for the soluble extracellular domain of murine VEGFR-3 (sR3-AP) was made using nucleotides 262363 of the murine VEGFR-3 (accession number L07296). sR3-AP was expressed and purified as reported for sR2-AP (20). In vitro binding and blocking assays were performed as described previously, except that murine sR3-AP or sR2-AP was used in place of human sR3-AP (16). Recombinant human VEGF-CN
C was used in all assays. The binding kinetics of sR3-AP or the mAb mF4-31C1 were measured by surface plasmon resonance on the BIACORE 2000 biosensor (BIACORE, Piscataway, NY). VEGF-C
N
C or soluble VEGFR-3 (sR3-AP) was immobilized on a sensor chip, and either sR3-AP or the mAb mF4-31C1 was injected over the surface of the sensor at various concentrations. Sensograms were evaluated using the BIA Evaluation 3.2 program to determine the binding rate constants.
Generation of Rat mAbs to Murine VEGFR-3
Lewis rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were primed with a subcutaneous injection of 100 mg of mR3-AP in complete Freunds adjuvant (Sigma). Rats received four intraperitoneal booster injections of 100 mg of mR3-AP at 2-week intervals. Rats whose sera showed the highest titer of inhibition in the VEGFR-3 blocking assay (see below) were injected intravenously with an additional 50 mg of sR3-AP. After 5 days, splenocytes were harvested and fused with mouse myeloma cells P3-X63-Ag8.653. Hybridomas were generated and subcloned according to standard protocols (21). Hybridomas secreting antibodies that bound to immobilized mR3-AP were further tested; anti-VEGFR-3 antibodies were selected based on positive binding to immobilized sR3-AP and further analyzed in the competitive VEGF-C blocking assay.
Receptor Phosphorylation
eEnd cells, an immortalized line of murine endothelial cells (a kind gift of Dr. Michael Pepper, University of Geneva Medical Center) (22) were serum starved overnight and incubated for 30 minutes in the presence or absence of mF4-31C1, nonimmune rat immunoglobulin G (IgG), or AFL4 prior to stimulation for 15 minutes with 100 ng/mL of either the 165-amino acid isoform of VEGF (VEGF165) (R&D Systems, Minneapolis, MN) or VEGF- CN
C. VEGFR-3 was immunoprecipitated from cell lysates using mF4-31C1 and protein G Sepharose resin (Amersham Biosciences, Uppsala, Sweden). Immunoprecipitated proteins were resolved by 4%20% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. Phosphotyrosine residues were detected by immunoblotting with the mAb PY-20 (Transduction Laboratories, Lexington, KY). Total VEGFR-3 was detected with a rabbit polyclonal antibody to mouse VEGFR-3 (M-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). mAb AFL4 was purchased from eBiosciences (San Diego, CA).
Mitogenic Assays with Cells Expressing Chimeric VEGFR-3-cFMS Receptor
cDNA encoding the extracellular domain of mouse VEGFR-3 was fused with cDNA encoding the transmembrane and cytoplasmic domains of the receptor for human colony-stimulating factor 1 (cFMS) in the expression vector pIres (Invitrogen). The DNA was electroporated into NIH-3T3 mouse fibroblast cells, and cell clones were selected by growth in G418. Plasma membrane expression of VEGFR-3-cFMS was demonstrated using indirect immunofluorescence with antibodies specific for murine VEGFR-3. Mitogenic assays were performed as described previously (16). Cells (5 x 103 per well) were plated onto 96-well tissue culture plates (Wallach, Inc., Gaithersburg, MD) and incubated in serum-free medium at 37°C for 72 hours. Various amounts of antibodies were added and preincubated at 37°C for 1 hour, after which VEGF-CN
C or VEGF165 was added to a 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 an additional 4 hours. The cells were placed on ice, washed once with serum-containing medium, incubated 10 minutes at 4°C with 10% tricholoroacetic acid, and solubilized in 25 µL of 2% sodiumdodecyl sulfate. Incorporated radioactivity was measured with a scintillation counter (Model 1450 Microbeta Scintillation Counter, Wallach).
Lymphangiogenesis Model
We recently developed a model of lymphangiogenesis in regenerating the tail 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 mice were used for each condition at each time point examined. Mice were anesthetized with a subcutaneous injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Postsurgical analgesic (buprenorphine, 2 mg/mL) was administered twice daily for 1 week by subcutaneous injection. All protocols were approved by the Animal Care and Use Committee of Northwestern University.
The regenerating region of skin was created as previously described (18). Briefly, a 2-mm-wide circumferential band of dermal tissue (in which the lymphatic network in the tail skin is contained) was excised midway up the tail, leaving the underlying bone, muscle, major blood vessels, and tendons intact. The area was then covered with a close-fitting, gas-permeable silicone sleeve and filled with type I rat tail collagen. The collagen provided a controlled environment in which skin could regenerate, and any lymphatic endothelial cells or structures later observed within this region were the result of newly initiated cell migration, proliferation, and organization.
Two variants of the model were used: 1) normal physiologic lymphatic regeneration in adult Balb/c mice and 2) lymphatic regeneration in the presence of excess tumor-derived VEGF-C in athymic mice. In the latter, VEGF-C-overexpressing or control-transfected human breast carcinoma cells (MDA-MB-435) (9) were implanted at 1 x 106 cells/mL within the collagen scaffold. Lymphatic regeneration was ascertained in Balb/c mice at 60 days and in tumor-bearing athymic mice at 25 days postsurgery (see below). In groups receiving mF4-31C1, the antibody was administered at 25 µg/g every 2 days by intraperitoneal injection beginning the day of surgery and proceeding until termination of the experiment.
Detection of Functional Lymphatic Vessels Via Microlymphangiography
To visualize lymph flow patterns both in situ as well as postfixation in thin sections, the animal was anesthetized and a 1% solution of tetramethylrhodamine isothiocyanate (TRITC)-conjugated, lysine-fixable dextran of 2 x 106 Da (Molecular Probes, Eugene, OR) was injected intradermally into the tail tip where it was taken up and transported by the lymphatic vessels in the proximal direction (23), revealing fluid channels and functional lymphatic vessels. The anesthetized animal was then killed with a perfusion through the blood vasculature with Zambonis fixative via the abdominal aorta (18). The tail was snap frozen in liquid nitrogen, stored at 80°C, and later cryosectioned. This fixation procedure resulted in crosslinking the dextran fluid tracer in place, thereby allowing lymph fluid to be visualized in cryosections and correlated to functional lymphatic vessels (e.g., dextran tracer colocalized with lymphatic endothelial cells).
Immunofluorescence and Immunohistochemistry
Tail specimens were cut into 10-µm longitudinal cryosections and immunostained. To detect lymphatic endothelial cells, a rabbit polyclonal antibody against the lymphatic-specific hyaluronan receptor LYVE-1 (24) (kind gift from Dr. David Jackson, John Radcliffe Hospital, Oxford, U.K.) was used along with a biotinylated goat anti-rabbit secondary antibody (Dako) and Alexa fluor 488-conjugated streptavidin (Molecular Probes). To detect blood endothelial cells, fluorescein isothiocyanate-conjugated anti-mouse monoclonal CD31 antibody (PharMingen) was used. Although lymphatic endothelial cells also express CD31, the expression is very weak compared with that of blood endothelial cells (18) and the two cell types could be readily differentiated based on staining intensity. As a negative control, normal skin was costained for CD31 and LYVE-1; no visible colocalization occurred (data not shown). Cell nuclei were labeled with 4',6-diamino-2-phenylindole (DAPI) (Vector Labs). To detect VEGF-C protein, sections were incubated with a goat anti-mouse antibody against VEGF-C (Santa Cruz Biotechnology) with a biotinylated rabbit anti-goat secondary antibody and Vector Red substrate (Vector Labs); nuclei were counterstained with hematoxylin.
Statistical Methods
At least three sections were counted per mouse with three to five mice per group. Data were presented as means with 95% confidence intervals (CIs). All P values were calculated using a two-sided Students t test.
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RESULTS |
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We began by generating a mAb to murine VEGFR-3. Hybridoma clones that produced antibodies with VEGFR-3 inhibitory activity were generated by fusing murine myeloma cells with splenocytes from a rat immunized with sR3-AP, a chimeric protein consisting of the soluble extracellular domain of murine VEGFR-3 fused to the human-secreted AP. Conditioned media from four clones inhibited 100%, 90%, 80%, and 50% of the binding of sR3-AP to immobilized VEGF-C. Stable monoclonal hybridoma cell lines mF4-31C1 and mF4-12A10 were established from the two clones with the highest blocking activity after subcloning three times. mF4-31C1 showed consistently more potent antagonist activity than mAb mF4-12A10 (data not shown) and was chosen for high-level production and purification. The isotype of mF4-31C1 was determined to be rat IgG 2a. The affinity constant of sR3-AP for the fully processed VEGF-CN
C was measured by BIACORE at 1 nM compared with 150 pM for the binding of mF4-31C1 to immobilized sR3-AP (Table 1).
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We utilized the immortalized murine endothelial cell line eEnd (22) to determine the capacity of mF4-31C1 to antagonize the VEGF-C-stimulated activation of VEGFR-3. Stimulation of serum-starved eEnd cells with recombinant human VEGF-CN
C resulted in the strong phosphorylation of VEGFR-3 (Fig. 2). In contrast, the addition of VEGF165 had no effect on VEGFR-3 phosphorylation. VEGF-C
N
C -mediated VEGFR-3 phosphorylation was blocked in a dose-dependent manner by mF4-31C1; mAb AFL4 had no effect (Fig. 2). The addition of mF4-31C1 to unstimulated cells did not phosphorylate VEGFR-3, even at the highest dose used (Fig. 2).
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The ability of mF4-31C1 to inhibit VEGF-CN
C -stimulated signal transduction was tested using an NIH-3T3 cell line that expresses VEGFR-3 fused to the transmembrane and cytoplasmic domains of the human receptor for colony-stimulating factor 1 (cFMS). Fluorescence-activated cell sorting analysis showed that the chimeric receptor was localized on the plasma membrane of transfected but not parental cells (data not shown). The incorporation of [3H]thymidine by the NIH-3T3 cells expressing VEGFR-3-cFMS was stimulated fourfold by the addition of VEGF-C
N
C but not VEGF165 (Fig. 3A). The mitogenic response was specifically blocked in a dose-dependent manner by mAb mF4-31C1 with a 50% inhibitory concentration of 1 nM (Fig. 3B). In contrast, mAb AFL4 did not inhibit the mitogenic response of cells expressing VEGFR-3-cFMS to VEGF-C
N
C at any concentration used.
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Having established mAb mF4-31C1 as a potent and unique antagonist of murine VEGFR-3 activation, we used this antibody to investigate the effects of VEGFR-3 inhibition on physiologically normal lymphangiogenesis. To this end, we used the mouse tail skin model of lymphangiogenesis in regenerating skin (18). Sixty days after collagen implantation, the lymphatic vessels of untreated mice had consistently and completely regenerated with nearly normal capillary architecture. Microlymphangiography showed that lymphatic transport was restored through the regenerated region, with continuity from the distal to proximal lymphatic capillary network (Fig. 4A). In a corresponding cryosection immunostained for LYVE-1, the location of lymphatic endothelial cells in the regenerating region overlapped with the location of lymph fluid tracer (i.e., TRITC-dextran), confirming that the lymphatic vessels in this region were functional (Fig. 4B). In contrast, in mice receiving mF4-31C1 by intraperitoneal injection over a 60-day period, lymphatic continuity was not restored (Fig. 4C) and lymphatic endothelial cells were rarely found within the regenerating region (Fig. 4D), although preexisting lymphatic vessels continued to function normally and none of the mice developed edema. Furthermore, the TRITC-dextran lymph tracer in the regenerating region did not overlap with lymphatic structures, indicating that lymph fluid was being transported mainly by interstitial convection through the region. Although the distal (upstream) and proximal (downstream) native lymphatic capillaries were disconnected by the regenerating region, the proximal vessels could be seen draining the lymph from the distal region, demonstrating the functional competency of both distal and proximal mature lymphatic vessels (Fig. 4, A and C).
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To determine blood endothelial cell density, cryosections were stained for CD31, an endothelial cell marker expressed prominently in blood endothelial cells (26), and strongly staining cells were counted. A slight reduction in blood endothelial cell density due to mF4-31C1 treatment was observed consistently, but the magnitude of this effect was small compared with the effect on lymphatic endothelial cells and failed to reach statistical significance in either Balb/c mice (P = .35, Fig. 7A) or athymic mice implanted with VEGF-C-overexpressing tumor cells (P = .38, Fig. 7B). Thus, inhibition of VEGFR-3 with the mAb mF4-31C1 did not substantially affect vascular angiogenesis in regenerating skin, even in the presence of excess tumor-derived VEGF-C.
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DISCUSSION |
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A surprising finding in the present report was that mAb AFL4 was not a potent antagonist of murine VEGFR-3. AFL4 has been shown to reduce lymphangiogenesis in a murine corneal pocket assay in which micropellets containing FGF-2 induced the production of VEGF-C primarily by blood endothelial cells (14), and it was also shown to inhibit tumor growth in several mouse tumor models (15). In the latter study, an important aspect of the antitumor effect of AFL4 was the disruption of the endothelial lining of postcapillary venules and microhemorrhage within the tumor tissue, suggesting a role for VEGFR-3 in stabilizing tumor vasculature. In view of our results on AFL4s ability to bind but not block VEGFR-3, it seems reasonable to suggest that the reported in vivo effects of AFL4 are mediated by a nonantagonist mechanism, such as the steric hindrance of VEGFR-3 dimerization or antibody-induced reduction in surface receptor expression, rather than by direct inhibition of ligand binding.
The mouse tail model of skin regeneration (17,18) has unique features which allowed us to incorporate VEGF-C-overexpressing tumor cells locally and to identify whether new lymphatic and blood vessels regenerated into the collagen scaffold that was initially devoid of vessels. Untreated, the excess tumor-derived VEGF-C led to grossly hyperplastic lymphatic vessels; this result is consistent with other reports of hyperplastic lymphatic vessels in transgenic mice overexpressing VEGF-C in the skin (27,28), in and around VEGF-C-overexpressing tumors (9,10,29), and following VEGF-C adenoviral expression (3032). However, we did not detect an increase in the number of lymphatic vessels in mice with VEGF-C overexpression (data not shown), even though it has been suggested that excess VEGF-C may also induce the growth of new lymphatic vessels when delivered by adenovirus (3032). Our finding in this study, that VEGFR-3 blocking completely prevented lymphatic regeneration in the presence of tumor-derived exogenous VEGF-C, is consistent with the notion that VEGFR-3 signaling is critical for tumor lymphangiogenesis.
Although lymphatic vessels completely failed to regenerate in the presence of mF4-31C1, we did not detect any differences in regenerating blood vessels or in preexisting lymphatic vessels (either in their appearance or in their ability to uptake and transport lymph), suggesting that the mF4-31C1 VEGFR-3 neutralizing antibody specifically blocks lymphatic regeneration. Other reports have suggested a role for VEGFR-3 signaling in stabilizing newly formed lymphatic vessels during embryonic development (13) as well as for lymphatic endothelial cell survival in vitro (12), implying that continuous VEGFR-3 signaling may be important for the survival or maintenance of existing lymphatic vessels. Our findings in adult tissues stand in contrast to these and suggest that VEGFR-3 signaling is not important for the survival of mature adult lymphatic vessels. Our findings further suggest that the role of VEGFR-3 signaling may be different in lymphatic development and adult regeneration. This finding is consistent with our recent observation that, although interstitial fluid flow was necessary for establishing normal lymphatic capillary organization in the regenerating skin, peak VEGF-C expression was seen only during the earliest stages of lymphangiogenesis (lymphatic endothelial cell proliferation and migration), with much less expression during the later stages of lymphatic capillary organization and functional integration (18).
In the present study, the initiation of both physiologically normal lymphangiogenesis and tumor lymphangiogenesis was prevented by the continuous neutralization of VEGFR-3, starting immediately upon initiation of skin regeneration (e.g., the implantation of the collagen scaffold into the mouse tail skin). In a clinical setting, however, tumor lymphangiogenesis may have already occurred by the time the tumor is identified. In order for anti-VEGFR-3 therapy to be effective in these patients, it must diminish existing tumor-associated lymphatic vessels. In contrast to normal functional lymphatic vessels, which are unaffected by VEGFR-3 blocking as demonstrated here, it is possible that tumor-associated lymphatic vessels may rely on continuous VEGF-C signaling for survival and hyperplasticity. However, it is currently unknown whether mF4-31C1 administration can reduce the density or size of existing hyperplastic lymphatic vessels in the tumor margin.
In summary, our observations in a unique model of adult lymphatic regeneration in mouse skin demonstrate that 1) VEGFR-3 signaling is necessary for the initiation of lymphatic regeneration, 2) VEGFR-3 signaling may not be required for the proper functioning of mature lymphatic vessels, and 3) VEGFR-3 neutralization can completely and specifically block adult lymphangiogenesis. These findings raise the possibility that human VEGFR-3 may be targeted therapeutically with antagonist mAbs to prevent the undesirable growth of lymphatic vessels, such as tumor-induced lymphatic hyperplasia or lymphangiogenesis.
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NOTES |
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The following authors are employees of ImClone Systems and may either hold stock or stock options in the company: B. Pytowski, K. Persaud, Y. Wu, L. Witte, and D. Hicklin. The results reported in this publication may impact decisions made by ImClone Systems regarding one of its potential products.
We thank Eva Mika, Seth ODay, Joe Rutkowski, Xenia Jimenez, and Laura Brennan for valuable assistance and Dr. David Jackson for the LYVE-1 antibody. We also gratefully acknowledge the support and advice of Dr. Peter Bohlen. We are grateful to the Illinois Division of the American Cancer Society, the Lurie Cancer Center of Northwestern University Medical School, and the NIH/NCI Breast Cancer SPORE (1 P50 CA89018-01) for their financial support of this project.
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Manuscript received August 4, 2004; revised October 15, 2003; accepted October 21, 2004.
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