Nonvascular VEGF receptor 3 expression by corneal
epithelium maintains avascularity and vision
Claus Cursiefen*†, Lu Chen*, Magali Saint-Geniez*, Pedram Hamrah*, Yiping Jin*, Saadia Rashid*, Bronislaw Pytowski‡,
Kris Persaud‡, Yan Wu‡, J. Wayne Streilein*§, and Reza Dana*¶
*Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street,
Boston, MA 02114;†Department of Ophthalmology, Friedrich-Alexander University Erlangen-Nu ¨rnberg, Schwabachanlage 6, 91054 Erlangen, Germany; and
‡ImClone Systems, Inc., 180 Varick Street, New York, NY 10014
Edited by Judah Folkman, Harvard Medical School, Boston, MA, and approved June 9, 2006 (received for review July 22, 2005)
Transparency of the cornea, the window of the eye, is a prereq-
uisite for vision. Angiogenesis into the normally avascular cornea
is incompatible with good vision and, therefore, the cornea is one
of the few tissues in the human body where avascularity is actively
maintained. Here, we provide evidence for a critical mechanism
contributing to corneal avascularity. VEGF receptor 3, normally
present on lymphatic and proliferating blood vascular endothe-
is mechanistically responsible for suppressing inflammatory cor-
angiogenesis ? cornea ? lymphatics ? inflammation
vascularity abruptly comes to an end at the cornea, the normally
as its main optical surface (Fig. 6, which is published as sup-
porting information on the PNAS web site). Indeed, corneal
species that require high visual acuity (1, 2). Conversely, blood
vessel growth into the cornea is incompatible with good vision
and is associated with the leading causes of corneal blindness
both worldwide (trachoma) and in industrialized nations (her-
Because of its lack of vascularity, the cornea has served as the
principal in vivo model system for studying vasculogenic processes,
specifically corneal hemangiogenesis (CHA) and more recently
lymphangiogenesis (3–8). However, the mechanisms underlying
maintenance of corneal avascularity remain poorly understood (2).
Several angiogenic growth factors, especially of the VEGF family,
have been implicated in mediating corneal angiogenesis (9, 10). As
thrombospondins 1 and 2, endostatin, pigment epithelium-derived
factor, and tissue inhibitor of metalloproteinases have been iden-
tified in the cornea (2). In addition, soluble VEGF receptor 1
corneal avascularity (11–13). However, to date, no single factor has
been identified as being critically responsible for maintaining
Recently, we observed that intact corneal epithelium can sup-
human corneal epithelial cells (15). Because VEGFR3 binds
VEGF-C and VEGF-D, and both of these factors promote lym-
phangiogenesis and hemangiogenesis and are additionally chemo-
tactic for inflammatory cells that secrete VEGF-A (8, 16), we
hypothesized that this ectopic VEGFR3 expression on the corneal
epithelium promotes avascularity of the normal cornea by serving
as a ‘‘sink’’ for VEGFR3 ligands.
he posterior structures of the eye, such as the choroid, are
among the most heavily vascularized tissues. Yet ocular
To test the hypothesis that VEGFR3 expression on the corneal
epithelium promotes avascularity of the normal cornea by
serving as a sink for VEGFR3 ligands, we first analyzed the
presence of VEGFR3 protein in murine corneal epithelial cells
(MCE) by using immunohistochemistry (Fig. 1A). Staining was
intense in the epithelial layer and only weakly positive in the
corneal endothelium and stroma. FACS analysis revealed strong
expression of VEGFR3 on corneal epithelial cells specifically
marked with the keratin-12 (K-12) marker (Fig. 1B). Strong gene
transcription of VEGFR3 in normal corneal epithelium was
confirmed by RT-PCR (Fig. 1C). Quantitative PCR demon-
strated higher expression levels of VEGFR3 in the corneal
epithelium compared with stroma?endothelium (1.5 times
higher; Fig. 1D). Finally, we determined whether VEGF-C can
indeed bind to ectopically expressed VEGFR3 on corneal epi-
thelial cells and whether it causes intracellular phosphorylation
events. First, we confirmed that MCE also express VEGFR3
(data not shown). Then, serum-starved MCE were treated with
VEGF-C, and VEGFR3 was immunoprecipitated with anti-
VEGFR3 antisera followed by Western blotting with antisera
against phosphotyrosine. Results revealed that VEGF-C can
bind to epithelial VEGFR3 and significantly increase the phos-
phorylation level of VEGFR3 (Fig. 1E). Surprisingly, there was
also some basal epithelial VEGFR3 phosphorylation even in the
absence of exogenous VEGF-C. These data demonstrate that
epithelial VEGFR3 is able to bind VEGF-C.
Next, to investigate a potential antiangiogenic role of corneal
that does not cause CHA in the presence of an intact corneal
epithelium (Fig. 2F); mild cautery of the cornea is known to
cause inflammatory cell influx into the cornea but fails to invoke
a neovascular response (ref. 17 and Fig. 2B). Similarly, removal
of the corneal epithelium alone (de-epithelialization) did not
cause CHA (Fig. 2C). In contrast, cauterization of de-
epithelialized corneas caused an angiogenic response, which by
morphometry was significantly greater compared with mice with
de-epithelialization alone (P ? 0.0001) or cautery alone (P ?
0.001), suggesting that intact corneal epithelium inhibits angio-
genesis (Fig. 2 E and F and Table 1).
To study whether corneal epithelium could inhibit inflamma-
tory cell influx into the cornea after a stimulus, the number of
inflammatory cells was compared at 72 h after cautery between
mice receiving cautery in the presence or absence of corneal
epithelium. There were significantly more inflammatory cells
corneas receiving cautery (352 ? 82 per section) compared with
the epithelialized corneas of mice that received cautery alone
(18 ? 6 per section; P ? 0.01).
Next, the established mouse model of suture-induced inflamma-
tory CHA was used (as a second in vivo model) to further study the
anti-inflammatory, antiangiogenic effects of corneal epithelium in
vivo. In this model three 11-0 sutures placed in the paracentral
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CHA, corneal hemangiogenesis; VEGFR, VEGF receptor; K-12, keratin-12;
MCE, murine corneal epithelial cells; CT, threshold cycle.
§Deceased March 15, 2004.
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
July 25, 2006 ?
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corneal stroma induced robust CHA within the first week postop-
eratively (ref. 8 and Fig. 3 A and B). We compared the degree of
CHA after suture placement in mice whose corneas were intact
versus those that were de-epithelialized. Absence of corneal epi-
thelium significantly enhanced the neovascular response compared
with corneas with intact epithelium (P ? 0.0001; Fig. 3E). Further-
more, sutures placed in corneas with intact corneal epithelium
displayed significantly reduced CD45 inflammatory cell recruit-
next layered corneal epithelium onto corneas that received central
sutures immediately after de-epithelialization and compared the
neovascular response with controls that were left de-epithelialized.
Morphometry of the area covered by blood vessels at day 7
demonstrated a significant inhibitory effect of corneal epithelium
on inflammatory CHA (32 ? 9.8%) compared with mice without
epithelial reapplication (51.8 ? 5.2%; P ? 0.001).
After confirming the significant suppressive effect of the epithe-
is mediated by VEGFR3–ligand interactions. First, we determined
whether an inflammatory angiogenic stimulus in the cornea is
associated with up-regulation of VEGFR3 ligands VEGF-C and
VEGF-D. Corneal suturing induced a significant (?3-fold) up-
regulation of both VEGF-C and VEFG-D mRNA (Fig. 3F),
whereas the VEGFR3 mRNA level remained unchanged (data not
Because the data described above established that (i) VEGFR3
is strongly expressed by the corneal epithelium, (ii) the epithelium
ectopic expression of both VEGFR3 protein (A and B) and mRNA (C and D) in
normal corneal epithelium. (A) Immunofluorescence at ?200. (Left) Anti-
VEGFR3 antibody. (Right) Control. Arrows indicate corneal epithelium. (B)
Two-color FACS staining against epithelial marker K-12 (phycoerythrin) and
VEGFR3 (FITC). Gray indicates isotype; black indicates VEGFR3 staining. Anal-
ysis was gated on K-12-phycoerythrin. Arrow indicates VEGFR3 and K12
costained corneal epithelium. (C) RT-PCR for VEGFR3 in normal corneal epi-
thelium (expected sizes: VEGFR3, 290 bp; GAPDH, 245 bp). (D) Quantitative
real-time PCR demonstrates higher levels of VEGFR3 in corneal epithelium
(Left) compared with stroma and corneal endothelium (Right). (E) VEGF-C
binds to corneal epithelial VEGFR3 and leads to VEGFR3 activation. Serum-
starved cultured MCE were treated with VEGF-C and VEGFR3 immunoprecipi-
tated from cell lysates by using a polyclonal anti-VEGFR3 antibody (M20) and
protein A-Sepharose. Immunoprecipitated proteins were resolved on an 8%
SDS?polyacrylamide gel and transferred to nitrocellulose membrane. Phos-
antibodies. After stripping the membrane, total VEGFR3 was detected by
using M20 antibody. The phosphorylation level of VEGFR3 was quantified by
densitometry and corrected to the amount of VEGFR3. A significant increase
in epithelial VEGFR3 phosphorylation after exposition to VEGF-C was shown.
Ectopic VEGFR3 expression in corneal epithelium. (A–D) Strong
Table 1. Summary of results from the experiment described
in Fig. 2F
conjunctival border. (B and D) A normally nonangiogenic inflammatory stim-
ulus (cautery; B) causes corneal neovascularization in the absence of epithe-
lium (D). (C) De-epithelialized cornea. Representative segments from corneal
flat mounts at the border between normally vascularized conjunctiva (Left)
and normally avascular cornea (Right) immunostained with CD31. (E) Mor-
phometry. (F) Diagram of experimental design. (Left) Cross section of normal
eye. (Right) Enlargement of cornea. Cells in corneal stroma are keratocytes;
red lines are invading blood vessels. (Magnification: A–D, ?100.)
Antiangiogenic effect of corneal epithelium I. (A) Normal corneal–
www.pnas.org?cgi?doi?10.1073?pnas.0506112103Cursiefen et al.
VEGFR3 ligands VEGF-C and VEGF–D are up-regulated in
conditions leading to inflammatory CHA, we next tested directly
whether epithelial VEGFR3 expression accounts for the antian-
vivo. We administered recombinant mouse VEGFR3?Fc chimeric
protein (ref. 18; comprising the amino acid residues 25–770 of the
extracellular domain of mouse VEGFR3 coupled to human Fc
IgG1) subconjunctivally to mouse eyes from which the corneal
epithelium had been removed and cautery applied. The Fc protein
served as control. Subconjunctival injection of neither Fc protein
whereas cauterized corneas that received control subconjunctival
injection displayed a strong angiogenic (Fig. 4) response after
de-epithelialization, cauterized deepithelialized corneas that were
treated with VEGFR3 chimeric protein displayed significantly
reduced CHA (P ? 0.0001), demonstrating that the constitutive
angiostatic effect of the corneal epithelium could be recreated in
the de-epithelialized cornea by a VEGFR3 chimeric molecule.
Next, we directly tested whether the antiangiogenic effect of
epithelial VEGFR3 could be suppressed in corneas receiving an
inflammatory stimulus. Syngeneic corneal epithelium was treated
30 min; 2.1 mg?ml) or control IgG, and then layered onto corneas
of BALB?c mice immediately after de-epithelialization. When
evaluated after 7 days (Fig. 5), ex vivo blockade of corneal epithe-
lium with anti-VEGFR3 significantly inhibited its antiangiogenic
capacity. Specifically, the vascularized area after corneal suturing
was significantly greater in the group receiving the anti-VEGFR3-
and direct support for VEGFR3-mediated (rather than another
epithelium-specific mechanism) suppression of CHA by corneal
The results provided here allow three important conclusions to
be drawn: First, corneal epithelial VEGFR3 expression and the
capacity to bind angiogenic growth factors VEGF-C and
VEGF-D constitutes a potent mechanism inhibiting inflamma-
tion-induced angiogenesis. Second, the data provide evidence
for an antiangiogenic role for VEGFR3. VEGFR3 may not only
provide proangiogenic signaling mediating hemangiogenesis and
lymphangiogenesis via ligation of VEGF-C and VEGF-D while
expressed on endothelium (as has been shown extensively; refs.
16 and 20–23), but VEGFR3 may also display antiangiogenic
properties when expressed at an avascular site by nonendothelial
(i.e., here epithelial) cells, where it can act as a decoy receptor.
Third, this regulation of corneal angiogenesis is critical for
maintenance of corneal clarity.
To date, no one factor has been identified as singularly critical to
maintaining corneal avascularity, a unique feature that the cornea
shares only with cartilage. The challenge of avascularity is partic-
ularly critical for the cornea, which because of its anatomically
exposed position and unkeratinized surface, is constantly con-
fronted with potential inflammatory stimuli such as particles land-
ing onto the eye surface and mechanical stresses from the micro-
abrasive effects of blinking and rubbing. The cornea therefore
needs to balance its usually high threshold for CHA with its ability
to react, when required, to sight-threatening injuries (e.g., aggres-
to enhance the immune defense against these threats (1). A system
that buffers low concentrations of angiogenic factors, but on the
other hand allows for angiogenesis to occur if a certain threshold is
reached, fits with the specificities of the VEGFR3 sink described
corneal epithelium (A) significantly enhances the neovascular response (ar-
rowhead: blood vessel) in the model of suture-induced inflammatory angio-
genesis [in parallel with increased influx of CD45?inflammatory cells (C and
D; CD45 immunostaining, arrows)] (B). (E) Results of morphometry. (F) Up-
regulation of VEGFR3 ligands in inflammatory corneal angiogenesis. The
ligands of VEGFR3, VEGF-C, and VEGF–D are significantly up-regulated in
conditions associated with inflammatory corneal angiogenesis (suture model
from B; P ? 0.05 for both ligands; lane 1, control; lane 2, suture-induced
inflammatory angiogenesis; expected sizes: VEGF-C, 531 bp; VEGF-D, 307 bp;
GAPDH, 245 bp). (Magnification: C and D, ?200.)
Angiosuppressive effect of corneal epithelium II. (A–D) Absence of
chimeric protein, ligating VEGF-C and VEGF-D, can substitute for the antian-
giogenic effect of VEGFR3-expressing corneal epithelium. The neovascular
response after cautery of de-epithelialized corneas (representative segment
Antiangiogenic effect of a VEGFR3 chimeric protein. A VEGFR3
Cursiefen et al.PNAS ?
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Why then is there not a ‘‘trap’’ for VEGF-A instead? VEGF-A,
the principal hemangiogenic growth factor binding to VEGFR1?2,
has been implicated as the key player in mediating CHA (3, 5, 10).
But there is no significant expression of VEGFR1?2 in the normal
cornea (11) and the physiologic VEGFR3 sink proposed herein is
unable to bind and neutralize VEGF-A. One likely explanation for
this seeming paradox is the fact that most of the minor insults to
which the cornea is constantly exposed, which would normally
cause unnecessary but potentially vision-threatening CHA, are
response that needs to be regulated is inflammatory-driven
explanations for this paradox exist; for example, it may well be that
the initial (e.g., IL-1 triggered) release of inflammatory VEGF-C
and VEGF-D is upstream of a subsequent VEGF-A release by
recruited neutrophils and macrophages (8). Therefore, an initial
(decoy receptor-mediated) neutralization of VEGF-C and
VEGF-D could interfere with this mechanism, that is, an initial
VEGF-C?-D sink might be enough to prevent the angiogenic
cascade if the causative stimulus is low intensity. According to this
concept, corneal epithelial VEGFR3 may be a physiologic trap to
prevent low-grade inflammation-induced, but potentially vision-
threatening and physiologically unnecessary, CHA. Additionally,
VEGFR3 signaling is important for maintenance of newly out-
grown blood vessels (25). Hence, in addition to VEGFR3 over-
expression by epithelium acting as a sink to deplete VEGFR2-
binding ligands VEGF-C and VEGF–D, corneal epithelial
VEGFR3 offers a mechanism to promote the regression of new
blood vessels if the angiogenic stimulus is not overwhelmingly
strong or sustained. Indeed, multiple and longstanding clinical and
animal experimental evidence relating persistent epithelial defects
in the setting of inflammation with potent CHA support this
concept of an epithelial ‘‘defense’’ against blood vessel in-growth.
Whereas several known inhibitors of angiogenesis have been
located within the cornea, the precise role of each of these factors
in maintaining corneal avascularity is unknown. The fact that
several inhibitors are present, and that genetic deletion of one or
several of them does not induce spontaneous corneal neovascular-
ization, suggests that a redundant system is maintaining corneal
avascularity. The fact that VEGF-C is the main angiogenic factor
up-regulated in an inflammatory milieu, and that it binds to its
ectopically expressed VEGFR3 on epithelial cells, suggests in the
aggregate that the VEGFR3 sink is important for maintaining
corneal avascularity in the setting of inflammation. Soluble
VEGFR1 binding VEGF-A in contrast may be more suitable for
blocking hypoxia-driven neovascularization.
VEGFR3 during development is expressed on venous and lym-
phatic vascular endothelium but later becomes largely restricted to
in the adult (16). For this reason it has commanded significant
interest because of its potential use in therapies for lymphatic
disorders and in oncology where tumor cell access to lymphatics is
critical for their metastasis (16, 26, 27). Interestingly, VEGFR3
knowledge in an adult tissue (28). This concept of an ‘‘ectopic’’
expression of a molecule that can in turn regulate a key tissue
response fits into a small, but growing, body of evidence that tissue
parenchymal cells may use cytokine and growth factor receptors as
sinks, or decoys, to provide an additional level of regulation for the
potentially pathological role of these factors (29). Because we
demonstrate that VEGF-C can ligate epithelial VEGFR3 and
induce receptor phosphorylation we cannot exclude an ‘‘antiangio-
sequestering bioactive ligand from endothelial VEGFR2?3). How-
ever, because a nonsignaling VEGFR3 chimeric protein in our
study could substitute for the antiangiogenic effect of corneal
epithelial VEGFR3 suggests that membrane-bound receptor sig-
naling is not essential for the antiangiogenic effect of this receptor
in the cornea, and that similar soluble receptors could be used
therapeutically to suppress pathological tissue responses.
The broad implications of our study are several-fold: First, these
data suggest a ‘‘check and balance’’ system involved in regulating
the angiogenic response to inflammation in a tissue whose avas-
cularity is critical for vision via constitutively high ectopic overex-
pression of VEGFR3 by the epithelium. Second, the data suggest
a dual and differential role of VEGFR3 in regulating angiogenesis
so that it can serve not only as a mediator but also as an inhibitor
of angiogenesis. Finally, these findings provide potential venues for
inhibition of sight-threatening corneal angiogenesis. Potential ther-
and secondary immune rejection or application of (cultured) cor-
neal epithelium onto common nonhealing corneal ulcers to pro-
mote wound healing without angiogenesis. Nonocular applications
could include induction of VEGFR3 overexpression by tumor
epithelia to suppress angiogenesis, just to name a few.
epithelial VEGFR3. Direct inhibition of epithelial VEGFR3 using ex vivo treat-
ment with neutralizing antibodies (19) diminishes the epithelium’s ability to
dampen angiogenesis. (A) Experimental design. (B and C) The neovascular
response to an inflammatory stimulus is significantly increased after ex vivo
treatment with a neutralizing anti-VEGFR3 antibody. Representative seg-
ments from CD31-stained corneal flat mounts (C) are compared with corneas
receiving epithelial transplants that received ex vivo treatment with control
IgG (B). (D) Morphometry. (Magnification: B and C, ?100.)
Antiangiogenic effect of corneal epithelium critically depends on
www.pnas.org?cgi?doi?10.1073?pnas.0506112103Cursiefen et al.
Materials and Methods
Mice and Anesthesia. BALB?c mice aged 6–8 weeks (Taconic
the Association for Research in Vision and Ophthalmology State-
ment for the Use of Animals. Mice were anesthetized with a
mixture of ketamine and xylazine (120 mg?kg body weight and 20
mg?kg body weight, respectively).
Epithelial Transplantation. Corneal de-epithelialization, cauterization,
and combined treatment. To induce an inflammatory but nonvascu-
larizing response in the cornea (17), a fine diathermy tip (Fine
Ophthalmic Tip, Aaron, St. Petersburg, FL) was placed briefly on
eight separate points for 1 s each within the central 2 mm of the
cornea. To examine the effect of the corneal epithelium on mod-
ulating CHA, the central 2 mm of the corneal surface was marked
the epithelium was removed with a microsurgical knife (5 mm;
Surgistar; Windsor Medical, Portsmouth, NH) by gently scraping
underlying corneal stroma. The completeness of de-epithelializa-
tion was verified by using hematoxylin?eosin serial sections and
fluorescein sodium staining in vivo (data not shown). Application
of cautery to these de-epithelialized corneas was performed to
determine whether the neovascular response had been altered.
Transplantation of corneal epithelial cell sheets. To further study the
was reapplied to the denuded area of the de-epithelialized eyes to
epithelium could be re-established. To accomplish this, the central
2 mm of syngeneic BALB?c donor corneas was excised and
incubated in 2% EDTA at 37°C for 1 h to enable separation of the
corneal epithelium from stroma as described (14). After three
washes with PBS for 5 min each, the epithelial sheet (referred to
subsequently as epithelium) was applied, basal layer down, to the
de-epithelialized surface of the recipient cornea. A surgical lid
closure was performed to secure the epithelium in place.
Mouse model of suture-induced, inflammatory corneal angiogenesis. The
mouse model of suture-induced inflammatory CHA (which unlike
cautery leads to profound CHA) was adopted as a contrasting
model to the cauterization model, which induces inflammation
without CHA (8). Briefly, a 2-mm corneal trephine was gently
placed on the cornea to mark the central corneal area. Three 11-0
sutures (Sharpoint Nylon 11-0; Surgical Specialty, Reading, PA)
were then placed intrastromally with two stromal incursions ex-
tending over 120° of corneal circumference each. The outer point
of suture placement was chosen as halfway between the limbus and
the line outlined by the 2-mm trephine; the inner suture point was
at the same distance from the 2-mm trephine line to obtain
standardized angiogenic responses. Sutures were left in place for 7
days. Mice were killed, and the cornea was then excised and a
flat-mount double-immunohistochemistry was performed as de-
scribed below (8).
Immunohistochemistry and Morphometry of Corneal Angiogenesis.
Briefly, corneal flat mounts were rinsed several times in PBS, fixed
in acetone, rinsed in PBS, blocked with 2% BSA, stained with an
FITC-conjugated CD31?PECAM-1 antibody overnight (1:100;
Santa Cruz Biotechnology), washed (three times for 5 min with
PBS), blocked, and stained with anti-LYVE-1 antibody overnight
(1:500; a lymphatic endothelium-specific hyaluronic acid receptor;
gift from D. Jackson, University of Oxford, Oxford, U.K.), which
was visualized with a Cy3-conjugated secondary antibody (1:100;
with a Zeiss Axiophot microscope. Digital pictures of the flat
mounts were taken with the Spot Image Analysis system, and the
area covered by CD31??LYVE-1?blood vessels (8) was measured
with National Institutes of Health IMAGE software. LYVE-1 stain-
vessels were measured and not CD31??LYVE-1?lymphatic ves-
sels. The total corneal area was outlined by using the innermost
vessel of the limbal arcade as the border, and the area of CHA was
as a percentage of the cornea covered by blood vessels). The mean
area of blood vessels extending beyond the limbus caused by the
from these values (i.e., 11%). Paraffin embedding of corneas and
immunostaining was done as described (15).
Immunohistochemistry for VEGFR3. To verify the expression of
VEGFR3 in MCE, immunohistochemistry on frozen and paraffin-
embedded sections of normal mouse eyes was performed with a
polyclonal rabbit–anti-mouse antibody (FLT-4, M20; 1:200; Santa
Cruz Biotechnology) and a polyclonal goat–anti-mouse antibody
(FLT-4, 102806 and 102804; 1:50; R & D Systems) as described (8,
15). EDTA treatment for 1 h leading to preparation of corneal
epithelial sheets did not reduce staining intensity for VEGFR3 on
Flow Cytometry. Epithelial cells of normal BALB?c mice were
obtained and used for flow analysis as described (30). Two-color
staining was used with the corneal epithelial-specific K-12 staining
red [phycoerythrin (PE); gift of Tung-Tien Sun, New York Uni-
versity Medical School, New York, NY] and the anti-VEGFR3
K-12?cells using appropriate isotype and cell culture controls to
adjust color compensation and gating parameters. Cells were
washed and analyzed with an Epics XL flow cytometer (Beckmann
Coulter). The proportion of K-12?cells that were also VEGFR3?
was quantified. As controls, we used rabbit IgG (for PE) and goat
IgG (for FITC; Santa Cruz Biotechnology).
Histological Quantification of Inflammatory Cells. The presence of
inflammatory cells in normal corneas and their recruitment into
corneas after microsurgical manipulations was quantified in hema-
toxylin?eosin-stained serial sections of plastic-embedded corneas,
fixed in 10% paraformaldehyde after enucleation. In addition,
corneal whole mounts and frozen sections were stained for the
Biotechnology), the panleukocyte marker CD45 (Pharmingen),
and the neutrophil marker GR1 (Pharmingen).
VEGFR3?Fc Chimeric Protein. To investigate whether exogenous
VEGFR3 inhibits inflammatory CHA and substitutes for the
putative function of corneal epithelial VEGFR3, we used a recom-
binant mouse VEGFR3 (FLT-4)?Fc chimeric protein (R & D
Systems) in mouse corneas subjected to de-epithelialization- and
cautery-induced CHA. For this, a DNA sequence encoding the
signal peptide from human CD33 joined with amino acid residues
25–770 of the extracellular domain of mouse VEGFR3 (18) was
fused to the 6? histidine-tagged Fc of human IgG1via a polypep-
tide linker. This chimeric protein was expressed in Sf21 cells. After
corneal de-epithelialization and cautery, one group of mice was
treated subconjunctivally with 10 ?l of VEGFR3 chimeric protein
immediately after surgery and 2, 4, and 6 days later. Control mice
received only the human IgG1-Fc instead (R & D Systems). The
degree of CHA was quantified 1 week after surgery and 24 h after
the last injection, using the above-described immunohistochemical
flat-mount morphometry. Each group comprised four mice, and
the experiment was performed twice with similar results.
Neutralizing Anti-VEGFR3 Antibody. A neutralizing anti-VEGFR3
antibody (mF4–31C1; ImClone Systems; ref. 19) was used to
directly test the role of corneal epithelial VEGFR3 expression on
angiogenesis. Using this anti-VEGFR3 antibody, we compared the
effect of epithelium incubated ex vivo in blocking anti-VEGFR3
antibody (2.1 mg?ml for 30 min) with the effect of similar epithe-
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at the same concentration. After extensive PBS washing, the
epithelium was layered onto inflamed and de-epithelialized cor-
neas, and the degree of CHA was measured.
RT-PCR for VEGF-C, VEGF-D, and VEGFR3 and Real-Time PCR for
VEGFR3. RT-PCR was carried out as described (2). Briefly, total
wood, TX). The central epithelium of corneas of 10 mice (20
corneas) was gently removed by scraping and then pooled
(similar results were obtained by pooling epithelium, which were
retrieved by 1 h of EDTA incubation; data not shown). In
addition, full-thickness corneas from 20 normal eyes and 20 eyes
bearing intrastromal sutures for 12 h were obtained to compare
VEGF-C and VEGF-D expression. From 1 ?g of mRNA, cDNA
was synthesized with Moloney murine leukemia virus reverse
transcriptase (Promega) according to the manufacturer’s in-
structions. The following primers were used for PCR from 5?
to 3?: GAPDH sense, GGTGAAGGTCGGTGTGAACGGA;
GAPDH antisense, TGTTAGTGGGGTCTCGCTCCTG;
sense, CGTTGCCTCATTGTGATTAG; VEGF-C sense,
GTCTGTGTCCAGCGTAGATG; VEGF-C antisense, GCTG-
TCTATGACA; and VEGF-D antisense, AGCACTTACAAC-
CCGTATGG. All primers were designed by Genosys (The
Woodlands, TX). PCR was carried out under the following
conditions: denaturation at 94°C, annealing at 55°C, and exten-
sion at 72°C. After 40 cycles of amplification (AmpliTaq DNA
polymerase; Applied Biosystems), PCR products were electro-
phoresed in 2% agarose gel and visualized by ethidium bromide
staining (0.5 ?g?ml ethidium bromide) for 40 min. Photographs
of the gel were taken with a high-resolution camera, and the
density of the bands was analyzed on the gel by using UV
illumination and IMAGE ONE image analysis software (Bio-Rad).
The expression level of mRNA was standardized by the expres-
sion of GAPDH as an internal control. The predicted sizes of
PCR products are 245 bp for GAPDH, 531 bp for VEGF-C, 307
bp for VEGF-D, and 290 bp for VEGFR3. For real-time PCR
for comparison of VEGFR3 levels in central corneal epithelium
versus central stroma and endothelium, first-strand cDNA was
synthesized from 1 ?g of total RNA with random hexamers by
using Super Script III (Invitrogen) according to the manufac-
turer’s protocol. Real-time PCR was performed with FAM-
MGB dye-labeled predesigned primers (Applied Biosystems) for
VEGFR3 (Assay ID Mm00433354?m1) according to the man-
ufacturer’s recommendations: 1 ?l of cDNA was loaded in each
well, and assays were performed in duplicate. A nontemplate
control was included in all of the experiments to evaluate DNA
contamination of the isolated RNA and the reagents used. The
comparative CT(threshold cycle) method was used to determine
the difference (?CT) between the CTof normal corneal stroma
CTwas normalized by the CTof the endogenous reference gene,
Immunoprecipitation and Western Blot Analysis. CulturedMCE(gift
of J. Niederkorn, University of Texas Southwestern Medical Cen-
ter, Dallas) were maintained in DMEM supplemented with 10%
FBS, 2 mM glutamine, and antibiotics at 37°C and CO2. To assay
VEGFR3 phosphorylation, subconfluent cells were incubated
overnight in serum-free media, incubated for 1 h in serum-free
media containing 100 ?M sodium orthovanadate, then stimulated
Reactions were terminated by washing in cold PBS. Cells were
collected in lysis buffer (10 mM Tris?HCl, pH 7.4?5 mM EDTA?50
mM NaCl?1% Triton X-100?50 mM NaF?1 mM PMSF?2 mM
A-Sepharose (Amersham Pharmacia Biosciences) for 1 h at 4°C.
VEGFR3 was immunoprecipitated by using a polyclonal rabbit
anti-mouse VEGFR3 antibody (M20; Santa Cruz Biotechnology)
overnight at 4°C. Immunoprecipitated complexes were analyzed by
SDS?PAGE using a mixture (1:1) of antiphosphotyrosine antibod-
ies: PY20 (Transduction Laboratories, Lexington, KY) and 4G10
(Upstate USA, Chicago). The membranes were stripped by incu-
bation for 30 min in 6.25 mM Tris?HCl (pH 6.8), 2% SDS, and 100
mM ?-mercaptoethanol at 50°C and reprobed with polyclonal
VEGFR3 antibody (M20) to detect total VEGFR3.
Whitney test. Differences were considered significant at P ?
0.05. Each experiment was performed at least twice with similar
results. Graphs were drawn with GraphPad (San Diego, CA)
PRISM, version 3.02.
and 47?1-2, the Interdisciplinary Center for Clinical Research (C.C.),
and National Institutes of Health Grants EY12963 (to R.D.) and
EY10765 (to J.W.S.).
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