a naturally occurring soluble form of vascular endothelial...

A Naturally Occurring Soluble Form of Vascular EndothelialGrowth Factor Receptor 2 Detected in Mouseand Human Plasma

John M.L. Ebos,1,2 Guido Bocci,1 Shan Man,1 Philip E. Thorpe,3 Daniel J. Hicklin,4 Danielle Zhou,5

Xiaohong Jia,5 and Robert S. Kerbel1,2

1Molecular and Cellular Biology Research, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario,Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 3Simmons ComprehensiveCancer Center, University of Texas Southwestern Medical Center, Dallas, Texas; 4ImClone Systems, New York, New Yorkand 5R&D Systems Inc., Minneapolis, Minnesota

AbstractAngiogenesis and vasculogenesis are regulated in large

part by several different growth factors and their

associated receptor tyrosine kinases (RTKs). Foremost

among these is the vascular endothelial growth factor

(VEGF) family including VEGF receptor (VEGFR)-2

and -1. VEGFR ligand binding and biological activity are

regulated at many levels, one of which is by a soluble,

circulating form of VEGFR-1 (sVEGFR-1). This sVEGFR-1

can act as a competitive inhibitor of its ligand, serve

as a possible biomarker, and play important roles in

cancer and other diseases such as preeclampsia.

Recombinant forms of sVEGFR-2 have been shown to

have antiangiogenic activity, but a naturally occurring

sVEGFR-2 has not been described previously. Here,

we report such an entity. Having a molecular weight

of f160 kDa, sVEGFR-2 can be detected in mouse and

human plasma with several different monoclonal and

polyclonal anti-VEGFR-2 antibodies using both ELISA

and immunoprecipitation techniques. In vitro studies

have determined that the sVEGFR-2 fragment can

be found in the conditioned media of mouse and

human endothelial cells, thus suggesting that it may

be secreted, similar to sVEGFR-1, or proteolytically

cleaved from the cell. Potential biological activity of this

protein was inferred from experiments in which mouse

sVEGFR-2 could bind to VEGF-coated plates. Similar

to sVEGFR-1 and other soluble circulating RTKs,

sVEGFR-2 may have regulatory consequences with

respect to VEGF-mediated angiogenesis as well

as potential to serve as a quantitative biomarker of

angiogenesis and antiangiogenic drug activity,

particularly for drugs that target VEGF or VEGFR-2.

(Mol Cancer Res 2004;2(6):315–26)

IntroductionAngiogenesis, the formation of new blood vessel capillaries

from an existing vasculature, and vasculogenesis, the de novo

formation of blood vessels from primitive precursor cells, are

dependent on several growth factors and their associated

tyrosine kinases. A key regulator of both these processes is

vascular endothelial growth factor (VEGF), also called vascular

permeability factor. Gene targeting methods have provided

powerful evidence for the indispensable role of VEGF signaling

systems in angiogenesis and vasculogenesis. For example,

disruption of only a single VEGF allele results in embryonic

lethality due to impaired vessel formation and function (1, 2).

Diseases involving angiogenesis such as cancer and age-related

vascular degeneration are often driven by VEGF, and as a

consequence, a variety of drugs, which target VEGF or VEGF

receptors (VEGFRs), are being developed and evaluated for

treatment of such diseases (3, 4). One such drug has already

been approved for the treatment of colorectal carcinoma

[i.e., bevacizumab, the humanized monoclonal anti-VEGF anti-

body (5)].

The VEGF family consists of VEGF-A (also called VEGF),

VEGF-B, VEGF-C, and VEGF-D as well as placental growth

factor (6). The most important of these in the context of tu-

mor angiogenesis is VEGF-A, especially the VEGF121 and

VEGF165 splice variant isoforms, which act both as an

activator and as a survival factor for endothelial cells of newly

formed blood vessels through binding to specific high-affinity

VEGFR tyrosine kinases. These receptors, known as VEGFR-2

or KDR (human)/flk-1 (mouse) and VEGFR-1 or flt-1, have

also been shown to be present on bone marrow–derived cells

(7), including hematopoietic stem cells (8) and circulating

endothelial progenitor cells (9); dendritic cells (10); trophoblast

and placenta cells (11); monocytes (12, 13); retinal progenitor

cells (14); and certain types of tumor (15). VEGFR-1 and -2

have an extracellular domain containing seven immunoglobu-

lin-like loops, a transmembrane domain, and an intracellular

split catalytic tyrosine kinase domain (16). Of the two receptors,

VEGFR-1 has very high affinity for VEGF binding (10-fold

Received 3/22/04; revised 5/17/04; accepted 5/20/04.Grant support: Ontario Graduate Scholarship in Science and Technology(J.M.L. Ebos); Sunnybrook Trust for Medical Research (G. Bocci); and NIH grantCA41223, Canadian Institutes for Health Research, National Cancer Institute ofCanada, and Canada Research Chair (R.S. Kerbel).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Robert S. Kerbel, Molecular and Cellular BiologyResearch, Sunnybrook and Women’s College Health Sciences Centre, S-217,2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. Phone: 416-480-5711; Fax: 416-480-5884. E-mail: [email protected] D 2004 American Association for Cancer Research.

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higher than VEGFR-2), but VEGFR-2, nevertheless, appears to

play a more important functional role in mediating signaling

events involved in endothelial cell mitogenesis, migration,

survival, and vascular permeability (17).

VEGFR-1 is not only found in a cell membrane–bound

form. Through alternative splicing of the VEGFR-1 mRNA, a

soluble, truncated form of VEGFR-1 (sVEGFR-1) is secreted

by endothelial cells (17-19). Comprised of six of the seven

immunoglobin-like domains present on the extracellular region,

this sVEGFR-1 can act as a natural endogenous inhibitor not

only by sequestering VEGF (20, 21) but also by binding and

inactivating membrane-bound VEGFR-1 and -2 receptors

through a mechanism involving a quasi form of receptor

homodimerization or heterodimerization (22). As such, the role

of sVEGFR-1 has been studied both as a surrogate marker for

disease progression and/or as a potential inhibitor of tumor

angiogenesis in cancers such as breast (23-25), ovarian (26),

colorectal carcinoma (27), renal cell carcinoma (28), fibroscar-

coma (29), and astrocytic tumors (30) as well as for its possible

contribution to diseases such as preeclampsia (31, 32). Of

interest in this regard is that many prior studies using synthetic

recombinant sVEGFR-2, encoding for the extracellular domain

of the full-length receptor, have shown similar characteristics to

that described for natural sVEGFR-1 such as the ability to bind

to VEGF (33-35) and VEGF/placental growth factor hetero-

dimers (36, 37) as well as mediating antitumor effects (38-40).

However, no natural form of sVEGFR-2 has been reported in

biological fluids.

The purpose of the present study was to investigate the pos-

sible presence of a sVEGFR-2 in mouse and human plasma. To

do so, we used eight different monoclonal and polyclonal an-

tibodies raised against the extracellular domain of mouse and

human VEGFR-2 and tested for the fragment by immunopre-

cipitation and ELISA both in vitro and in vivo. Our results show

that sVEGFR-2 can indeed be detected in mouse and human

plasma, the functional significance of which is unknown.

ResultsImmunoprecipitation of a sVEGFR-2 Fragment FromMouse Plasma Using Anti-VEGFR-2 Antibodies

A spliced variant of VEGFR-2 mRNA, encoding for all but

a portion of the intracellular catalytic domain, was described

previously by Wen et al. (41) in rat retinal cells; however,

there have been no reports of a naturally occurring truncated

VEGFR-2 protein found in biological fluids. Putative

sVEGFR-2 was immunoprecipitated from severe combined

immunodeficiency (SCID) mouse plasma using protein

G–coated Sepharose beads incubated with one of three

different rat-derived monoclonal anti-mouse antibodies raised

against the extracellular domain of mouse VEGFR-2. The

antibodies used—DC101 and RAFL-1/RAFL-2—were raised

independently against recombinant sVEGFR-2 and were

derived by different techniques. Immunoblotting using a

polyclonal anti-mouse anti-VEGFR-2 antibody revealed anf160-kDa protein for each sVEGFR-2 immunoprecipitation

performed (Fig. 1A, lanes 1 , 3 , and 5). Recombinant

sVEGFR-2, designated (M)-sVEGFR-2/Fc, which encodes for

amino acid residues 1 to 762 of the extracellular domain of

mouse VEGFR-2, was used for comparison (Fig. 1A, lane 8).

sVEGFR-2 Depletion After ImmunoprecipitationConfirmed by Mouse sVEGFR-2 ELISA

Supernatants from each immunoprecipitation shown in

Fig. 1A were tested for levels of sVEGFR-2 depletion using a

mouse sVEGFR-2 ELISA. Results show that the postimmuno-

precipitation supernatants contained less sVEGFR-2 (Fig. 1B,

lanes 1 , 3 , and 5) corresponding to 0, 7.36, and 3.49 pg/mL,

respectively) when compared with the control sample of plas-

ma alone (Fig. 1B, lane 7 corresponding to 40.26 pg/mL),

thus confirming that this mouse sVEGFR-2 ELISA is mea-

suring the same sVEGFR-2 fragment immunoprecipitated in

Fig. 1A as well as indicating that each of the antibodies used

are binding the same protein.

Plasma-Derived Mouse sVEGFR-2 Binds to VEGFThe same samples used in the above experiment (post-

immunoprecipitation supernatants) were incubated in parallel

on 96-well plates coated with 4 Ag per well recombinant mouse

VEGF to test whether this sVEGFR-2 fragment found in the

plasma could bind VEGF (Fig. 1C). Control sample, containing

plasma alone, incubated on the VEGF-coated plate (Fig. 1C,

lane 7 , 22.6 pg/mL) showed sVEGFR-2 levels similar to levels

measured by mouse sVEGFR-2 ELISA (Fig. 1B, lane 7 , 40.26

pg/mL). Additionally, as in Fig. 1B, each sample was compared

with control sample of plasma alone (Fig. 1B, lanes 1 to 6),

confirming again that immunoprecipitation supernatants were

depleted of sVEGFR-2. Recombinant mouse (M)-sVEGFR-2/

Fc was used to generate standard curves for both sVEGFR-2

ELISA and VEGF-coated plates (Fig. 1D). Curves show that

recombinant (M)-sVEGFR-2/Fc is less sensitive at binding

the VEGF-coated plate compared with the mouse sVEGFR-2

ELISA, potentially explaining why levels of sVEGFR-2

(Fig. 1B, lane 7 and Fig. 1C, lane 7) differed marginally

between the two detection methods.

Plasma-Derived Mouse sVEGFR-2 Is HeavilyGlycosylated

It has been demonstrated previously that recombinant

sVEGFR-2, derived from insect cells, has a molecular weight

of f100 to 110 kDa (34, 35). Additionally, because the cDNA-

derived amino acid sequence of sVEGFR-2 has 16 potential

N-linked glycosylation sites, Huang et al. (34) further showed

that this insect-derived recombinant sVEGFR-2, when degly-

cosylated, is f80 to 90 kDa. We hypothesized that the reas-

on the f160-kDa mouse plasma-derived sVEGFR-2 we report

has slower mobility on SDS-PAGE than the previously re-

ported insect-derived recombinant proteins is due to the

more extensive glycosylation that occurs in mammalian cells

(42). To test this, sVEGFR-2 was immunoprecipitated from

SCID mouse plasma using DC101 and R&D antibodies

(Fig. 2, lanes 1 and 3, respectively) and treated with peptide

N-glycosidase (PNGase) to remove all N-linked glycoproteins.

After treatment, sVEGFR-2 was reduced from f160 to f90

kDa (Fig. 2, lanes 2 and 4 , respectively) by SDS-PAGE analysis,

indicating that the mammalian-derived sVEGFR-2 is more

heavily glycosylated than the insect-derived sVEGFR-2 and,

when deglycosylated, is of comparable size with the previ-

ously reported deglycosylated insect-derived recombinant

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FIGURE 1. Immunoprecip-itation of a naturally occurringsVEGFR-2 from SCID mouseplasma. A. A sVEGFR-2(f160-kDa) fragment was im-munoprecipitated from SCIDmouse plasma using one ofthree different rat-derivedmonoclonal antibodies specificfor mouse VEGFR-2. Antibod-ies used were DC101,RAFL-1,or RAFL-2. Controls includedantibody incubated alone withbeads, plasma incubatedalone with beads, and re-combinant extracellular do-main of mouse VEGFR-2fused to a human Fc [desig-nated (M)-sVEGFR-2/Fc]. B.Post immunoprec ip i ta t ionsupernatants were tested onsVEGFR-2 ELISA and foundto be depleted of sVEGFR-2,indicating that the proteinmeasured by this ELISA isthe same as that immuno-precipitated in A. C. Usingthe same samples as in B,parallel experiments were per-formed using VEGF-coatedplates (4 Ag per well) andfound to yield a similar result,suggesting potential biologicalactivity of this sVEGFR-2 pro-tein. D. Values for B and Cwere derived from curves us-ing recombinant (M)-sVEGFR-2/Fc as a standard.

Naturally Occuring sVEGFR-2


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sVEGFR-2. To further illustrate this difference in glycosylation,

mammalian-derived sVEGFR-2 [designated (M)-sVEGFR-2/Fc]

and insect-derived recombinant sVEGFR-2 [designated

(I)-sVEGFR-2] were compared after treatment with PNGase.

(M)-sVEGFR-2/Fc is an f180-kDa protein and becomes 120

kDa when deglycosylated (Fig. 2, lanes 5 and 6 , respectively),

while the (I)-sVEGFR-2 is f110 to 115 kDa and becomesf90 to 95 kDa when deglycosylated (Fig. 2, lanes 7 and 8 ,

respectively). We attribute the fact that the (M)-sVEGFR-2/Fc

is larger than both the glycosylated and the deglycosylated

mouse-derived sVEGFR-2 to be due to the human Fc IgG1

portion to which the recombinant is fused (see Materials and

Methods for description).

Conditioned Media From Mouse Endothelial CellsContains sVEGFR-2

Conditioned media (CM) taken from endothelial cells of

human origin have been shown to contain sVEGFR-1 (20, 21).

Using VEGFR-2 (+) mouse endothelial cell lines PY4.1, MS1,

and bEnd3, we assessed the levels of sVEGFR-2 detectable in

CM. Immunoprecipitations were performed using both CM and

cell lysates (which would contain the full-length VEGFR-2)

with the mouse monoclonal VEGFR-2 antibody DC101 or

RAFL-2. Immunoblotting using a polyclonal anti-VEGFR-2

antibody revealed a full-length 180- to 250-kDa VEGFR-2

protein in the total cell lysates of PY4.1 (Fig. 3A, lane 8), MS1

(Fig. 3C, lane 6), and bEnd3 (Fig. 3C, lane 13) as well as thef160-kDa sVEGFR-2 protein found in the CM (Fig. 3A, lanes

4 and 6 and Fig. 3C, lanes 4 and 11 for PY4.1, MS1, and

bEnd3 cells, respectively). Aliquots of supernatants were taken

from before and after immunoprecipitation and tested for

sVEGFR-2 depletion by mouse sVEGFR-2 ELISA to confirm

that the immunoprecipitated protein is the same as measured in

the mouse sVEGFR-2 ELISA kit. CM from PY4.1, MS1, and

bEnd3 taken after immunoprecipitation (Fig. 3B, lanes 4 and 6

and Fig. 3D, lanes 4 and 11 , respectively) contained lower

levels of sVEGFR-2 than in CM taken from before immuno-

precipitation (Fig. 3B, lane 2 and Fig. 3D, lanes 2 and 9 ,

respectively). Control media showed no detectable levels of

sVEGFR-2 by immunoprecipitation (Fig. 3A, lanes 3 and 5

and Fig. 3C, lanes 3 and 10, respectively) or by mouse

sVEGFR-2 ELISA (Fig. 3B, lanes 1 , 3 , and 5 and Fig. 3D,

lanes 1 , 3 , 8 , and 10 , respectively). Similarly, VEGFR-2 was

detected by sVEGFR-2 ELISA in PY4.1, MS1, and bEnd3

cell lysates (Fig. 3B, lane 7 and Fig. 3D, lanes 5 and 12,

respectively) and shown to be depleted in supernatants after

immunoprecipitation (Fig. 3B, lane 8 and Fig. 3D, lanes 6 and

13 , respectively). Finally, postimmunoprecipitation superna-

tants from both MS1 and bEnd3 CM were incubated in parallel

on 96-well plates coated with 4 Ag per well recombinant

mouse VEGF to test whether the sVEGFR-2 fragment detected

could bind VEGF (Fig. 3E). CM of MS1 and bEnd3 taken after

immunoprecipitation (Fig. 3E, lanes 4 and 11 , respectively)

contained lower levels of sVEGFR-2 than in CM taken from

before immunoprecipitation (Fig. 3E, lanes 2 and 9 , respec-

tively), confirming again that both immunoprecipitation super-

natants were depleted of sVEGFR-2 and that the sVEGFR-2

detected by the VEGF-coated plates is the same protein

FIGURE 2. Deglycosyla-tion of sVEGFR-2 and recom-binant sVEGFR-2 proteinsincreases mobility on SDS-PAGE gel. The f160-kDanaturally occurring sVEGFR-2protein, immunoprecipitatedfrom SCID mouse plasmausing DC101 or R&D antibod-ies (lanes 1 and 3), migratedon SDS-PAGE gel at f90kDa after deglycosylation withPNGase F (lanes 2 and 4).Differences in glycosylationbetween two recombinantsVEGFR-2 proteins, onemammalian derived [designat-ed (M)-sVEGFR-2/Fc; lane 5 ]and one insect derived [desig-nated (I)-sVEGFR-2; lane 7 ],were compared. Mobility of(M)-sVEGFR-2/Fc and (I)-sVEGFR-2 by SDS-PAGEanalysis was increased tof120 and 90 to 95 kDa,respectively (lanes 6 and 8),after deglycosylation withPNGase F, indicating thatthe mammal ian -de r i vedsVEGFR-2 is more heavilyglycosylated than the insect-derived protein.

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measured by the sVEGFR-2 ELISA. VEGFR-2 and sVEGFR-2

could not be detected in lysates or CM of VEGFR-2 (�) celllines such as KS.1, HB60, and CB357 (data not shown). Taken

together, these results demonstrate that the sVEGFR-2 protein

immunoprecipitated from the CM and plasma and the VEGFR-

2 immunoprecipitated from the cell lysate are the same

sVEGFR-2 and VEGFR-2 protein measured by the mouse

sVEGFR-2 ELISA.

Human Plasma and Endothelial Cell CM ContainsVEGFR-2

Using experimental protocols identical to the studies we

undertook using mouse samples, sVEGFR-2 was immunopre-

cipitated from both human plasma and human VEGFR-2 (+)

endothelial cell CM using the highly specific phage display–

derived monoclonal antibody, 1121, raised against the extracel-

lular domain of human VEGFR-2. The f160-kDa sVEGFR-2

protein, revealed by immunoblotting using a horseradish

peroxidase–conjugated polyclonal anti-VEGFR-2 antibody,

was identified from two human plasma samples taken from

healthy volunteers (Fig. 4A, lanes 2 and 4) and compared with

the full-length 180- to 250-kDa VEGFR-2 protein detected

from cell lysates of human endothelial cell lines, human

umbilical vascular endothelial cell (HUVEC) and EA.hy926

(Fig. 4A, lanes 6 and 8 , respectively). Levels of VEGFR-2

on EA.hy926 cells were markedly lower than on HUVECs.

Immunoprecipitation controls using plasma or cell lysate alone

(Fig. 4A, lanes 1 , 3 , 5 , and 7) showed no protein, while a

control with 1121 antibody alone (Fig. 4A, lane 9) revealed

partially unreduced 1121 antibody which cross-reacted with the

anti-VEGFR-2-horseradish peroxidase antibody used for im-

munoblotting. Using a sandwich ELISA for human sVEGFR-2,

postimmunoprecipitation supernatants of plasma-alone controls

showed sVEGFR-2 in both plasma samples (Fig. 4B, lanes 1

and 3) and VEGFR-2 in both cell lysate controls (Fig. 4B, lanes

5 and 7). In contrast, supernatants after immunoprecipitation

with 1121 were shown to be depleted of sVEGFR-2 (Fig. 4B,

lanes 2 and 4) and VEGFR-2 (Fig. 4B, lanes 6 and 8), con-

firming that the full-length VEGFR-2 protein and its soluble

truncated form, detected by immunoprecipitation, is the same as

that detected by ELISA.

Additionally, as was shown previously for mouse endothe-

lial cell CM, sVEGFR-2 could be immunoprecipitated from

HUVEC CM using the 1121 antibody (Fig. 4C, lane 4),

whereas control media contained no such protein (Fig. 4C,

lane 3). Human sVEGFR-2 ELISA results showed that

postimmunoprecipitation CM (Fig. 4D, lane 4) had lower

sVEGFR-2 than before immunoprecipitation (Fig. 4D, lane 2).

Taken together, these results confirm that the protein detected in

human plasma and human endothelial cell CM, measured by

both immunoprecipitation and ELISA, is the same full-length

VEGFR-2 protein or sVEGFR-2 fragment.

sVEGFR-2 Levels in Plasma and Serum of HealthyVolunteers

From eight healthy volunteers (4 male and 4 female), blood

was collected and the concentration of sVEGFR-2 was

measured in serum, heparin plasma, EDTA plasma, and citrate

plasma by sVEGFR-2 ELISA. Table 1 summarizes the normal

values of sVEGFR-2 tested in all materials. No statistical

differences (P > 0.05) were found either between the sVEGFR-

2 levels in the serum and plasma types or between the male

and female groups as determined by the Mann-Whitney test

(GraphPad Prism Software Package version 4.0). Furthermore,

linear regression analysis was performed to compare the

sVEGFR-2 levels in the serum and the plasma samples

obtained from the same healthy volunteer. A high correlation

(P < 0.0001) between sVEGFR-2 levels of serum and the

three types of plasma samples of the same volunteer was

found, suggesting that sVEGFR-2 levels could potentially be

measured accurately using either method (data not shown).

However, before the normal levels of sVEGFR-2 in healthy

volunteers can be precisely defined, further measurements will

need to be performed with a larger population group.

DiscussionOur results demonstrate for the first time that a sVEGFR-2

can be detected in plasma. Five different monoclonal anti-

bodies, raised against the extracellular domain of either human

or mouse VEGFR-2, were used to precipitate the f160-kDa

sVEGFR-2 fragment from normal human or SCID mouse

plasma. The anti-VEGFR-2 antibodies used included the

mouse VEGFR-2 (flk-1) specific DC101, R&D, and RAFL-1/

RAFL-2 antibodies as well as the human VEGFR-2 (KDR)

specific antibody, 1121, all of which were generated inde-

pendently. Our studies confirmed that the detected soluble

fragment was a truncated form of VEGFR-2 by comparison

with the full-length 180- to 250-kDa VEGFR-2, precipitated

from mouse and human endothelial cell lysates, and the f180-

kDa recombinant mouse sVEGFR-2 [(M)-sVEGFR-2/Fc],

containing the extracellular domain fused to a human Fc.

Further studies also revealed that the f160-kDa mouse-

derived sVEGFR-2 protein is heavily glycosylated as cleavage

of N-linked glycoproteins with PNGase resulted in sVEGFR-2

migration at 90 kDa on SDS-PAGE gel. Using two human and

mouse polyclonal anti-VEGFR-2 antibodies, our findings

show that both the immunoprecipitated natural sVEGFR-2

and the full-length VEGFR-2, as well as mammalian- and

insect-derived recombinant sVEGFR-2, could be distinguished

by Western blot analysis.

Because endothelial cells of human origin are known to

secrete sVEGFR-1 (20, 21), we asked whether a sVEGFR-2

could be detected in the CM of various endothelial cell lines of

both mouse and human origin. Our results, using three mouse

and two human VEGFR-2–positive endothelial cell lines

demonstrate that the f160-kDa protein, identical in size to

the sVEGFR-2 fragment precipitated from plasma, could be

detected in the CM using the aforementioned immunoprecip-

itation technique. This suggests that, like sVEGFR-1, one

source for sVEGFR-2 detected in the plasma might be

endothelial cell in origin. For VEGFR-1, the secretion of the

soluble fragment is a consequence of alternative mRNA

splicing. While Wen et al. (41) have demonstrated that

alternative splicing of VEGFR-2 mRNA in rat retinal cells

can result in a truncated form of VEGFR-2 encoding for a

functional form of receptor lacking a portion of the intracellular



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domain, there have been no reports of sVEGFR-2 arising as a

result of alternative mRNA splicing. Whether the sVEGFR-2

we detect in the plasma and CM of endothelial cells is a result

of alternative mRNA splicing, proteolytic cleavage of the

membrane-bound receptor, or another mechanism, is still

unknown and currently under investigation.

An important aspect of our study was that the sVEGFR-2

detected in the plasma and CM could be confirmed using

commercially available sVEGFR-2 sandwich ELISA kits

designed to capture and detect the extracellular domain of both

human and murine VEGFR-2 using monoclonal and polyclonal

antibodies. Using aliquots of supernatants taken from before

and after the immunoprecipitation of sVEGFR-2 from both

plasma and CM, we found that detectable levels of sVEGFR-2

were either markedly reduced or depleted entirely. This result

was an important confirmation that the f160-kDa sVEGFR-2

fragment, identified from the aforementioned immunoprecipi-

tations, was the same sVEGFR-2 fragment measured by both

human and mouse ELISA kits. Because our findings demon-

strate that one possible source of sVEGFR-2 might be

endothelial cells, this result raises the possibility that detection

of natural sVEGFR-2 in the plasma using such ELISA methods

FIGURE 3. Immunoprecipitation of a naturally occurring sVEGFR-2 from CM of mouse endothelial cells. A. From confluent plates, PY4.1 mouseendothelial cell lysates and CM were tested for VEGFR-2 and sVEGFR-2 levels, respectively. Immunoprecipitations were performed from media and celllysates using DC101 or RAFL-2. CM contained the same f160-kDa sVEGFR-2 fragment shown present in plasma (lanes 4 and 6), while cell lysate revealedthe full-length 180- to 250-kDa VEGFR-2 (lane 8 ). Recombinant murine (M)-sVEGFR-2/Fc is shown for size comparison (lane 9 ). B. For media and celllysates, aliquots of preimmunoprecipitation and postimmunoprecipitation supernatants were saved for sVEGFR-2 and VEGFR-2 level comparison.Postimmunoprecipitation supernatant showed depletion of sVEGFR-2 and VEGFR-2 as measured by mouse sVEGFR-2 ELISA. Using the identical protocolas above, experiments with MS1 and bEnd3 yielded the same results by immunoprecipitation and Western blotting (C) and depletion of sVEGFR-2 andVEGFR-2 from postimmunoprecipitation supernatants (D). Parallel experiments performed using the same immunoprecipitation supernatants used in Dshowed that sVEGFR-2 derived from MS1 and bEnd3 could bind to VEGF-coated plates (4 Ag per well) (E) in a similar manner to the mouse sVEGFR-2 ELISA.+, either cell lysate or CM; �, either control media or lysis buffer; *, lysates were not tested on VEGF-coated plate.

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may have potential use in the evaluation of sVEGFR-2 in

preclinical or clinical samples. Because other soluble receptor

forms, such as soluble c-kit in germ cell tumors (43), sTie2

and VEGFR-1 in renal cancers (44), and sHER-2/neu in

metastatic breast cancers (45), have been studied as potential

surrogate markers for disease progression, and the fact that

elevated VEGFR-2 expression has been shown in some

diseases to be correlated to tumor progression (46), it is

plausible that circulating sVEGFR-2 levels might be similarly

useful as an indicator for tumor progression. Experiments to

determine whether circulating sVEGFR-2 levels might be

used as such a surrogate marker for tumor progression and/

or response to certain antiangiogenic drugs are currently

under way.

FIGURE 3 continued.



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Synthetic recombinant forms of both mouse and human

sVEGFR-2, encoding for the extracellular region of the receptor,

have been used previously for two main purposes—(1) as an

immunogen for development of anti-VEGFR-2 antibodies

(including those used in this study) and (2) as a potential anti-

angiogenic agent both in vitro (34-37, 39) and in vivo (38, 47,

48)—because recombinant sVEGFR-2 retains ligand binding

ability and can therefore reduce bioavailability of VEGF. For

this reason, we asked whether the natural form of sVEGFR-2

could bind to VEGF in a similar fashion. Using plates coated

with recombinant murine VEGF, we tested whether sVEGFR-

2 derived from both SCID mouse plasma and mouse

endothelial cell CM could be captured in a manner identical

to that used in the sVEGFR-2 ELISA studies. Experiments

using the murine sVEGFR-2 ELISA and VEGF-coated plates

in parallel demonstrated that the sVEGFR-2 detected in both

SCID mouse plasma and mouse endothelial cell CM yield

similar results. This finding suggests that, like sVEGFR-1,

sVEGFR-2 may have biological activity. Whether this nat-

ural sVEGFR-2 can play a significant role in VEGF sig-

naling, as has been described for sVEGFR-1, remains to be

established.

Finally, an important point to note is that all monoclonal

antibodies used for the immunoprecipitation of sVEGFR-2 from

the plasma and CM, with the exception of R&D, were developed

to inhibit VEGF binding to VEGFR-2 (49-51). This raises the

possibility that these antibodies, shown to mediate antiangio-

genic effects, could potentially bind sVEGFR-2 in vivo. Because

it is unknownwhether sVEGFR-2 is involved in VEGF signaling

or if the fragment contributes any significant biological role in

angiogenesis, an intriguing possibility to consider is that

sVEGFR-2 may influence (or be influenced by) the activity of

drugs that are designed to block VEGFR-2. In addition, it may

have potential as an indicator for drug efficacy and/or optimal

biological dosing, at least for agents that target VEGFR-2 and

perhaps other angiogenesis inhibitors.

FIGURE 4. sVEGFR-2 detected in human plasma and CM from human endothelial cells. A. Immunoprecipitations of sVEGFR-2 from two human plasmasamples and VEGFR-2 from HUVEC and EA.hy926 cell lysates were performed using the human anti-VEGFR-2 antibody 1121 raised against the extracellulardomain of VEGFR-2. B. Postimmunoprecipitation supernatants showed marked depletion of sVEGFR-2 and VEGFR-2 compared with plasma-alone or celllysate – alone controls. C. Similar studies revealed sVEGFR-2 in the CM of HUVEC. D. Postimmunoprecipitation supernatants tested by human sVEGFR-2ELISA proved to be depleted of sVEGFR-2, further confirming that the f160-kDa sVEGFR-2 and full-length 180 to 250-kDa VEGFR-2 measured by Westernblotting and by ELISA in both plasma and CM are the same protein. +, cell lysate, plasma, or CM; �, either control media or lysis buffer.

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Materials and MethodsAntibodies

Several different anti-VEGFR-2 antibodies, raised against

the extracellular region of mouse and human recombinant

VEGFR-2, were used to identify the full-length VEGFR-2 and

sVEGFR-2 fragment by immunoprecipitation. For studies

using mouse samples, the rat-derived mouse monoclonal anti-

VEGFR-2 antibodies used were DC101 (ImClone Systems,

New York, NY; ref. 49), RAFL-1 and RAFL-2 (kind gifts from

Dr. Philip E. Thorpe, University of Texas, Dallas, TX; ref. 50),

and R&D (R&D Systems, Inc., Minneapolis, MN; clone

91292.11). For human samples, the phage display–derived

FIGURE 4 continued.

Table 1. Detection of sVEGFR-2 in the Plasma or Serum of Normal Healthy Human Volunteers

sVEGFR-2 (pg/mL)

Plasma Heparin Plasma EDTA Plasma Citrate Serum

All healthy volunteers* (n = 8) Range 5,511-10,499 5,339-9,961 4,704-8,262 5,493-10,945Mean F SD 8,216 F 1,537 8,264 F 1,525 7,098 F 1,235 8,469 F 1,64795% Confidence interval 6,932-9,501 6,989-9,539 6,066-8,130 7,092-9,845

Female (n = 4) Range 5,511-9,086 5,339-9,166 4,704-8,262 5,493-9,509Mean F SD 7,341 F 1,463 7,397 F 1,601 6,470 F 1,461 7,601 F 1,653

Male (n = 4) Range 7,741-10,499 7,816-9,961 6,815-8,096 7,902-10,945Mean F SD 9,091 F 1,152 9,131 F 927.3 7,726 F 609.2 9,336 F 1,261

NOTE: sVEGFR-2 levels in serum, heparin plasma, EDTA plasma, and citrate plasma of healthy volunteers (total volunteers = 8; 4 female and 4 male). sVEGFR-2 levelswere defined by human sVEGFR-2 ELISA.*age range: 22-31.



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monoclonal antibody 1121 (ImClone Systems; ref. 51) was

used. Detection of VEGFR-2 and sVEGFR-2 in Western

immunoblotting was performed using goat-derived mouse and

human specific anti-VEGFR-2 antibodies conjugated to

horseradish peroxidase (R&D Systems).

Cell LinesTransformed VEGFR-2 (+) mouse endothelial cell lines

used were PY4.1 (derived from s.c. microvessels of SV40T

transgenic mice; ref. 52), MS1 (immortalized with a temper-

ature-sensitive SV40 large T antigen; ref. 53), and bEnd3

(originally derived from mouse brain capillaries; ref. 54). All

murine cell lines used were generous gifts of Dr. Daniel J.

Dumont (Sunnybrook and Women’s College Health Sciences

Centre, Toronto, Ontario, Canada). Murine VEGFR-2 (�) celllines used as negative controls were HB60, KS.1, and CB357

(55). Human umbilical vein endothelial cells used included

both a primary cell line (HUVECs; American Tissue Culture

Collection, Manassas, VA; CRL-1730) and an immortalized

cell line (EA.hy926; ref. 56). HUVECs were maintained as

described previously (57). All other cells were routinely cul-

tured in DMEM supplemented with 5% FCS. Cell cultures

were incubated at 37jC and 5% CO2 in a humidified incubator.

Recombinant Mouse sVEGFR-2Two recombinant mouse sVEGFR-2 proteins were used

in this study, one derived from mammalian cells, designated

(M)-sVEGFR-2/Fc, and the other from insect cells, designated

(I)-sVEGFR-2. Both recombinant proteins encode for amino

acid residues 1 to 762 of the extracellular domain of mouse

VEGFR-2 (58). (M)-sVEGFR-2/Fc was obtained from R&D

Systems (catalogue 443-KD), is used as the standard for the

mouse sVEGFR-2 ELISA kit, and was fused to the 6� histidine-

tagged Fc of human IgG1 via the peptide (IEGRMD) resulting

in a disulfide-linked homodimeric protein, containing two

986–amino acid residue subunits. The chimeric protein was

expressed in a mouse myeloma cell line, NS0. (I)-sVEGFR-2

(a kind gift of Dr. Thorpe) was derived from the insect cell

Spodoptera frugiperda (Sf9) infected with sVEGFR-2 recom-

binant baculovirus as described previously (34).

Plasma CollectionPlasma samples from SCID mice (Taconic Laboratories,

Germantown, NY) were obtained after anesthesia with

isofluorane, and mice were bled from the retroorbital sinus or

by cardiac puncture. The blood samples were collected in

Microtainer (Becton Dickinson, Franklin Lakes, NJ) plasma-

separating tubes and centrifuged at 4jC. Plasma was immedi-

ately pooled, frozen, and stored at �70jC until assayed. All

capillary tubing, syringes, and needles used for bleeding were

first rinsed with heparin to avoid any clotting. Human venous

blood samples from healthy volunteers were obtained using a

21-gauge needle, with blood aliquoted into Vacutainer (Becton

Dickinson) bottles and then centrifuged to collect serum (BD

catalogue 367986), sodium heparin plasma (BD catalogue

367874), K2 EDTA plasma (BD catalogue 367861), and

sodium citrate plasma (BD catalogue 363083). All samples

were frozen and thawed at 4jC immediately before immuno-

precipitation or ELISA analysis.

Collection and Concentration of Endothelial Cell CMAll endothelial cell lines grown to f80% confluency were

incubated for 48 hours with control media incubated in parallel.

After collection, CM and control media were centrifuged at

1000 rpm for 10 minutes, debris was removed, and supernatant

was filtered through 0.44 Am filters (Millipore, Billerica, MA).

Both control media and CM were placed in a cold speedvac

(Savant, Albertville, MN) and concentrated to maximize the

concentration of sVEGFR-2. Samples reduced by 85% of their

original volume, for the both control media and CM, were

pooled and used to test sVEGFR-2 expression by immunopre-

cipitation and ELISA studies.

ImmunoprecipitationStep 1.

Plasma or Endothelial Cell CM ‘‘Clearing.’’ Protein G

Sepharose 4 Fast Flow beads (Pharmacia Biotech, Uppsala,

Sweden) were washed three times in lysis buffer [20 mmol/L

Tris (pH 7.5), 137 mmol/L NaCl, 100 mmol/L NaF, 10%

glycerol, 1% NP40, and 1 mmol/L Na2VO4] to remove ethanol

storage solution. Pooled SCID plasma samples were then

thawed and incubated at 4jC for 2 to 12 hours with the protein

G beads (100 AL beads per 1 mL plasma) on a rotator. Plasma

was removed from beads by centrifugation, and preclearing

procedure was repeated twice more. Beads used for these

preclearing steps were saved and later analyzed by Western

blotting to show removal of proteins binding nonspecifically to

the beads but not removal of sVEGFR-2 (data not shown).

Preclearing procedure was also performed for both the mouse

endothelial cell CM and control media (both reduced by 85%

from their original volume).

VEGFR-2 Antibody Preincubation. Protein G–coated

Sepharose beads (100 AL) were incubated for 24 hours with

100 Ag of VEGFR-2 antibody in lysis buffer. Antibody-bound

beads were then centrifuged and washed once in lysis buffer

before adding the precleared plasma.

Step 2.

Immunoprecipitations for sVEGFR-2From‘‘Cleared’’Plasma

and CM. Precleared plasma was quantified by Bradford assay

(Bio-Rad, Hercules, CA), and 10 mg of protein were added to

antibody-bound beads (see Step 1) and filled to a volume of 1

mL with lysis buffer. After 24 hours of rotating at 4jC, beadswere centrifuged and supernatants were saved for later analysis

by sVEGFR-2 ELISA. Beads were washed three times in cold

lysis buffer and resuspended in loading buffer and a reducing

agent (10% 1,4-DTT; Boehringer Mannheim, Laval, Quebec,

Canada). Beadswere boiled at 100jC to remove protein-antibody

complexes, and samples were analyzed by Western blot.

The identical protocol was performed for immunoprecipita-

tions from endothelial cell CM (reduced by 85% original vol-

ume), with the exception of one modification—precleared

endothelial cell CM was added directly to the beads contain-

ing antibody, in equal volumes, and no dilution was made. Endo-

thelial cell control media was incubated in parallel in all cases.

Deglycosylation of sVEGFR-2To assess and compare glycosylation of the sVEGFR-2

protein, protein G–coated Sepharose beads containing the

Ebos et al.


324



sVEGFR-2-antibody complexes (see above immunoprecipita-

tion protocol) or the recombinant sVEGFR-2 protein were

boiled in glycoprotein denaturing buffer and incubated with

PNGase F (New England Biolabs, Beverly, MA; catalogue

P0704S) for 1 hour at 37jC as per manufacturer’s instructions

prior to SDS-PAGE analysis.

Western BlottingProteins were resolved using 7.5% SDS-PAGE gels and

blotted onto polyvinylidene difluoride membranes (Immobilon-P,

Millipore, Nepean, Ontario, Canada) by electrophoretic trans-

fer (Hoefer Pharmacia Biotech, San Francisco, CA). Prior to

immunoblotting, membranes were blocked in 10% nonfat

milk in TBS-T buffer [10 mmol/L Tris (pH 7.5), 150 mmol/L

NaCl, and 0.1% Tween 20] and subjected to immunoblot

analysis as described previously (59).

Measurement of sVEGFR-2 Protein Level by ELISAHuman and Murine sVEGFR-2 ELISA. Levels of sVEGFR-2

were assessed using a commercially available mouse and

human sVEGFR-2 sandwich ELISA assays (R&D Systems,

catalogue MVR200 and DVR200) following manufacturer’s

instructions. Immunoprecipitation supernatants from control

media and CM were added, with no dilution, to the ELISA to

maximize low signals. All other samples were diluted as per

manufacturer’s instructions. Both ELISA kits are composed of

a monoclonal capture antibody and horseradish peroxidase–

conjugated polyclonal antibody for detection. Both human and

mouse polyclonal antibodies were used for immunoblotting,

and the mouse monoclonal (designated ‘‘R&D’’ above) was

used for immunoprecipitation studies.

VEGF-Coated Plates. Plates (96 wells) were coated with

4 Ag/mL recombinant mouse VEGF165 at 150 AL per well

overnight at room temperature. Plates were then washed twice

in wash buffer and blocked overnight with 350 AL per well of a

bovine serum albumin–based blocking buffer. On the third day,

plates were aspirated and dried in a vacuum oven at 25jC to

30jC. VEGF-coated plates were analyzed using the identical

protocol as the mouse sVEGFR-2 ELISA using the same

reagents (see above).

AcknowledgmentsWe thank Cassandra Cheng for her excellent administrative assistance; Dr. GuilioFrancia, Dr. Urban Emmenegger, Mariano Loza-Coll, Alexandra Haninec, andJeanne du Manoir for their advice and critical review of this manuscript; Dr.Daniel J. Dumont for the MS1, bEnd3, and PY4.1 cells; Dr. David E. Spaner forhelp with human sample collection; and Dr. Sophia Ran for help in the productionof the RAFL-1 and RAFL-2.

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2004;2:315-326. Mol Cancer Res John M.L. Ebos, Guido Bocci, Shan Man, et al. of Canada (R.S. Kerbel).Institutes for Health Research, and National Cancer Institute Research (G. Bocci); and NIH grant CA41223, CanadianTechnology (J.M.L. Ebos); Sunnybrook Trust for Medical

Ontario Graduate Scholarship in Science and11PlasmaGrowth Factor Receptor 2 Detected in Mouse and Human A Naturally Occurring Soluble Form of Vascular Endothelial

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