The role of different VEGF isoforms in scar formation after glaucoma

filtration surgeryq

Tine Van Bergena,*, Evelien Vandewallea,b, Sara Van de Veirea,b,c, Mieke Dewerchinc,Jean-Marie Stassend, Lieve Moonse, Ingeborg Stalmansa,b,*

a Laboratory of Ophthalmology, KU Leuven, Herestraat 49, 3000 Leuven, BelgiumbDepartment of Ophthalmology, University Hospitals Leuven, Kapucijnenvoer 33, 3000 Leuven, BelgiumcVesalius Research Center, VIB e KU Leuven, Herestraat 49, 3000 Leuven, Belgiumd ThromboGenics NV, Gaston Geenslaan 1, 3001 Heverlee, BelgiumeDepartment of Biology, KU Leuven, Naamsestraat 61, 3000 Leuven, Belgium

a r t i c l e i n f o

Article history:

Received 23 May 2011

Accepted in revised form 29 August 2011

Available online 1 September 2011

Keywords:

vascular endothelial growth factor

pegaptanib

Tenon fibroblasts

human umbilical vein endothelial cells

glaucoma filtration surgery

wound healing

a b s t r a c t

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological

angiogenesis. Our previous studies showed a differential role of VEGF isoforms in retinal physiological

angiogenesis. We also demonstrated that non-selective inhibition of VEGF by bevacizumab had a bene-

ficial effect on surgical outcome after glaucoma filtration surgery by reducing angiogenesis as well as

fibrosis. However, the function of the VEGF isoforms in pathological angiogenesis and wound healing in

the eye still remains unidentified. This study was designed to elucidate the differential roles of VEGF

isoforms in scar formation after trabeculectomy. Furthermore, we also investigated whether pegaptanib

(Macugen�, Pfizer), an aptamer which specifically blocks VEGF165, could improve surgical outcome by

reducing postoperative scarring. VEGF-R2 and neuropilin-1 (NRP-1) expression was analyzed in vitro by

RT-PCR, and were found to be expressed at higher levels in human umbilical vein endothelial cells

(HUVEC) as compared to Tenon fibroblasts (TF). The effect of the different VEGF isoforms (VEGF121,

VEGF165 and VEGF189) and pegaptanib on cell proliferation was determined via WST-1 assay. Endothelial

cell proliferation was stimulated after addition of VEGF121 and VEGF165, whereas VEGF121 and VEGF189increased fibroblast growth. These effects on proliferation were associated with an activation of the ERK

pathway, as revealed using the TransAM c-Myc assay. Inhibition of the ERK pathway, by PD98059

administration, significantly reduced VEGF isoform induced cell growth. A dose-dependent reduction of

endothelial cell proliferation was observed after pegaptanib administration, while only the highest dose

was able to inhibit fibroblast growth. Next, the in vivo effect of pegaptanib was investigated in a rabbit

model of trabeculectomy. The surgical outcome was evaluated by performing clinical investigations (IOP,

bleb area, height and survival), as well as histomorphometric analyses of angiogenesis (CD31), inflam-

mation (CD45) and fibrosis (Sirius Red). A single postoperative application of pegaptanib had a beneficial

impact on surgical outcome, mainly by reducing angiogenesis, but not inflammation or collagen depo-

sition. Repeated injections slightly improved surgical outcome, but again solely by reducing angiogen-

esis. In summary, our results revealed that the VEGF isoforms play a differential role in ocular wound

healing: VEGF165 and VEGF121 predominantly affect blood vessel growth, whereas VEGF189 is rather

involved in fibrosis, an important process in wound healing.

� 2011 Elsevier Ltd. All rights reserved.

Abbreviations: VEGF, vascular endothelial growth factor; VEGF-R2, VEGF receptor 2; NRP-1, neuropilin-1; HSPGs, heparin sulphate proteoglycans; HUVEC, human

umbilical vein endothelial cells; HTF, human Tenon fibroblasts; RTF, rabbit Tenon fibroblast.q Supported by IWT (Agency for Innovation by Science and Technology in Flanders), FWO (Research Foundation Flanders) and FRO (Funds for Research in Ophthalmology).

* Corresponding authors. Laboratory of Ophthalmology, KU Leuven, Herestraat 49, Box 1025, B-3000 Leuven, Belgium. Tel.: þ32 16 33 04 61; fax: þ32 16 33 04 67.

E-mail addresses: tine.vanbergen@med.kuleuven.be (T. Van Bergen), evelien.vandewalle@med.kuleuven.be (E. Vandewalle), sara.vandeveire@uzleuven.be (S. Van de Veire),

mieke.dewerchin@vib-kuleuven.be (M. Dewerchin), jmstassen@thrombogenics.com (J.-M. Stassen), lieve.moons@bio.kuleuven.be (L. Moons), ingeborg.stalmans@uzleuven.be

(I. Stalmans).

Contents lists available at SciVerse ScienceDirect

Experimental Eye Research

journal homepage: www.elsevier .com/locate/yexer

0014-4835/$ e see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.exer.2011.08.016

Experimental Eye Research 93 (2011) 689e699

1. Introduction

Vascular endothelial growth factor (VEGF) is best known as an

endothelial and permeability factor and plays a role in both normal

(e.g. embryonic development) (Carmeliet et al.,1996) and pathological

angiogenesis (e.g. in tumour growth, inflammation, wound healing

and various ocular diseases) (Dvorak et al., 1999; Kowanetz and

Ferrara, 2006; Wilgus et al., 2008). VEGF is known to stimulate

endothelial cells (Rini and Small, 2005) and many non-vascular cells,

such as Tenon fibroblasts (Li et al., 2009). It acts primarily via three

high-affinity VEGF tyrosine kinase receptors: Flt-1 (VEGF-R1), KDR

(VEGF-R2) and Flt-4 (VEGF-R3) (Ferrara et al., 2003; Li et al., 2009).

VEGF-R2 mediates most biologically relevant VEGF responses,

including cell migration and proliferation (Zachary, 2003). VEGF

binding to this receptor mediates the activation of the mitogen-

activated protein kinase kinase (MEK)/extracellular signal-regulated

kinase (ERK) cascade, known to be a major mediator of VEGF-

induced proliferation, both in endothelial cells (Takahashi et al.,

2001) and fibroblasts (Wu et al., 2004). Upon activation, ERK phos-

phorylates a variety of intracellular targets, one of which is c-Myc,

a short-lived transcription factor involved in multiple aspects of

growth control (Seth et al., 1992) and proliferation (Pap et al., 2004;

Pintus et al., 2002).

Alternative splicing of a single VEGF-gene results in multiple

isoforms, such as VEGF121, VEGF165 and VEGF189. These isoforms

are classified by the number of amino acids (Zhang et al., 2008)

and differ in molecular mass, solubility and (co-)receptor binding

properties (Stalmans et al., 2002). Although it is well-known that

the different isoforms bind VEGF-R2 with equal affinity (Yamazaki

and Morita, 2006; Zhang et al., 2000), they do differ in their

affinity for VEGF-R co-receptors, such as neuropilin-1 (NRP-1) and

heparin sulphate proteoglycans (HSPGs). The presence of

sequences encoded by exon 6 or 7 regulates interactions with

these co-receptors (Olsson et al., 2006; Robinson and Stringer,

2001). VEGF121 lacks these exons and therefore also lacks the

HSPGs- and NRP-1-binding domains. NRP-1, recognizing the exon-

7-encoded domain of VEGF, acts as a co-receptor for VEGF165 and

VEGF189 and enhances their binding to VEGF-R2 and their bioac-

tivity (Soker et al., 2002). HSPGs, known to be expressed on the

cell surface of almost all mammalian cells, interact with many

proteins. VEGF165, lacking exon 6, has a moderate affinity for

HSPGs, while VEGF189 contains an additional sequence encoded by

exon 6a and therefore strongly binds HSPGs (Olsson et al., 2006;

Robinson and Stringer, 2001).

The differential role of the VEGF isoforms in vascular patterning

and arterial development in the retina has been described

(Stalmans et al., 2002). However, the function of these isoforms in

pathological angiogenesis and wound healing in the eye still

remains unidentified. Recently, we showed that VEGF plays an

important role in scar formation after filtration surgery. VEGF was

expressed in samples of aqueous humor from glaucoma patients

and rabbits that had undergone filtration surgery. Furthermore,

VEGF stimulated the proliferation of Tenon fibroblasts in vitro and

different isoforms (VEGF121, VEGF165, VEGF189) were expressed by

these cells (Li et al., 2009). Recently, Zhang et al. (Zhang et al., 2008)

reported different vascular responses to VEGF121 and VEGF165.

While VEGF165 significantly stimulated the signalling pathway in

human umbilical vein endothelial cells (HUVEC), VEGF121 had little

effect. These data suggest that VEGF isoforms might play distinct

roles by activating different signalling pathways.

Pegaptanib sodium (Macugen�, Pfizer, New York), an aptamer

that prevents the binding of VEGF165 to its receptor, has been

approved by the Food and Drugs Administration for the treatment

of neovascular age-relatedmacular degeneration (AMD). The phase

III VEGF Inhibition Study in Ocular Neovascularization (VISION)

clinical trials showed that intravitreal injections of pegaptanib slow

visual loss in neovascular AMD (Ciulla and Rosenfeld, 2009).

The aim of this study was to elucidate the role of the VEGF

isoforms in scar formation after glaucoma filtration surgery.

Different in vitro and in vivo models were used to study angio-

genesis, as well as inflammatory and fibrotic processes. Secondly,

we evaluated the therapeutic potential of selective VEGF inhibition

(pegaptanib) on ocular wound healing, using a rabbit model of

glaucoma filtration surgery.

2. Materials and methods

All animal procedures were performed in accordance with the

ARVO Statement for the Use of Animals in Ophthalmic and Vision

Research and the EC Directive 86/609/EEC for animal experiments,

using the guidelines of our own institution.

2.1. Cells and culture conditions

Human umbilical vein endothelial cells (HUVEC) (Lonza, Wal-

kersville, Maryland) and Tenon fibroblasts (TF) were used. Human

Tenon fibroblasts (HTF) were obtained during strabismus surgery

and rabbit Tenon fibroblasts (RTF) were harvested during filtration

surgery, as previously described (Li et al., 2009).

HUVEC and TF were maintained at 37 �C in a humidified

atmosphere of 5% CO2 in respectively EGM-2 and DMEM complete

medium, as previously described (Li et al., 2009). EGM-2 medium

was supplemented with 10% foetal bovine serum (FBS), 0.1% human

recombinant epidermal growth factor, 0.4% recombinant human

fibroblast growth factor, 0.1% VEGF, 0.1% recombinant lung insulin-

like growth factor, 0.04% hydrocortisone, 0.1% ascorbic acid, 0.1%

heparin, 0.1% gentamicin sulphate amphotericin-B (EGM-2 Bullet-

Kit, Lonza, Walkersville, Maryland). HUVEC were cultured in 0.1%

gelatine (Invitrogen, Carlsbad, California) coated dishes. In all

experiments cells between the 3rd and 6th passage were used.

2.2. Real-time RT-PCR

To investigate the expression of VEGF-R2 and NRP-1 at the

mRNA level, RNA from HTF and HUVEC was isolated using the

RNeasy Minikit (Qiagen, Valencia, California), as described previ-

ously (Carmeliet et al., 2001). Expression was normalized to that

of the housekeeping gene b-actin. Primers and probes, designed

using Primer Express� 10 software, were described previously

(Brusselmans et al., 2005; Li et al., 2009).

2.3. Proliferation assay

To investigate the effect of the different VEGF isoforms and

pegaptanib, HUVEC, HTF and RTF were seeded in (0.1% gelatine

coated) 96-well plates (Falcon, Becton Dickinson Labware, Franklin

Lakes, New Jersey) at an initial density of 5 � 103 cells per well in

100 ml complete medium. In one series of experiments (6 h after

seeding), the cells were serum starved (medium supplementedwith

0.1% FBS) overnight. The medium was changed to fresh serum-free

medium containing 10, 25 and 100 ng/ml or 0.25, 1, 2, 5, 10 nM

recombinant human VEGF121 (4644-VS; R&D Systems, Minneapolis,

Minnesota), VEGF165 (293-VE; R&D Systems, Minneapolis, Minne-

sota) or VEGF189 (CRV114B, Cell Sciences, Canton, Massachusetts).

Forty-eight hours after administration, cell proliferation was

assessed using the WST-1 Cell Proliferation Assay System (Roche,

Mannheim, Germany). To examine whether inhibition of the ERK

pathway would exert any influence on proliferation of HTF and

HUVEC, cellswere pretreatedwith 20 mMPD98059 (an ERK pathway

inhibitor) 2 h prior to VEGF-treatments (25 ng/ml). Three hours after

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699690

VEGFadministration, cell proliferationwas assessed using theWST-1

Cell Proliferation Assay. Different time periods and concentrations

were chosen based on a study by Zhang et al. (Zhang et al., 2008),

shown to exert inhibitory effects on HUVEC proliferation.

In a second series, the medium was replaced by complete

medium, pre-incubated with different concentrations of pegapta-

nib (0, 0.03, 0.3, 1 and 2 mg/ml) or 10 mg/ml of bevacizumab, an

anti-VEGF antibody, known to exert pronounced inhibitory effects

on TF proliferation (Li et al., 2009). Of note, the pegaptanib

concentrations were based on those that are routinely used in

clinical practice for intravitreal injections, the highest concentra-

tion being the one that could be obtained by minimal dilution with

the required culture medium. Forty-eight hours after administra-

tion, cell proliferation was assessed using the WST-1 Cell Prolifer-

ation Assay. Complete or serum-free medium served as control.

2.4. Transcription factor activation assay

To clarify the differential effects elicited by VEGF121, VEGF165 and

VEGF189 on HUVEC and HTF, cells were serum starved overnight and

treated with the different isoforms (25 ng/ml) for 10 min, a concen-

tration and time period shown to exert the highest increase in acti-

vation of the MEK/ERK pathway in HUVEC (Zhang et al., 2008).

Serum-freemedium served as control. Nuclear protein extracts were

obtained from both cell types using a Nuclear Extract Kit (40010;

Active Motif, Rixensart, Belgium), as previously described (Akiyama

et al., 2004). Shortly, the cells were collected in ice-cold PBS in the

presence of phosphatase inhibitors. After resuspension in hypotonic

buffer, detergent was added to collect the cytoplasmic fraction.

Nuclei were lysed and nuclear proteins were solubilised in lysis

buffer. The protein concentration of the nuclear extract was

measured by Pierce bicinchoninic acid (BCA) protein assay kit

(23225, Thermo Scientific, Rockford, Illinois). ERK DNA binding

activity was assessed by the TransAM c-Myc assay, according the

manufacturer’s instructions (43396; Active Motif, Rixensart, Bel-

gium). The nuclear extract (5 mg) was subjected to binding of c-Myc

using an immobilized consensus sequence in a 96-well plate, and

primary and secondary antibodies were added. The colorimetric

reaction was analyzed using a spectrophotometer and DNA binding

specificitywas assessedusingmutated orwild-type oligonucleotides

(20 pmol/well). The wild-type consensus oligonucleotide prevented

c-Myc binding to the probe immobilized on the plate. Conversely, the

mutated consensus oligonucleotide had no effect on c-Myc binding.

2.5. Rabbit model of filtration surgery

Female New Zealand rabbits, 12e14 weeks of age and weighing

2e3 kg, were used. Before surgery, the intra-ocular pressure (IOP)

wasmeasured in both eyes with a tonometer (Tono-Pen;Medtronic

Solan, Jacksonville, Florida) under 4 mg/ml topical anaesthesia

(Unicain, Thea Pharma, Schaffhausen, Switzerland). Three record-

ings per eye were averaged. General anaesthesia was induced by

intramuscular injection of 50 mg/ml ketamine (Ketalar, Pfizer, Ann

Arbor, Michigan) and 2% sedative (Rompun, Bayer Health Care,

Pittsburgh, Pennsylvania). Surgery was performed on both eyes,

using a technique as previously described (Khaw et al., 1993).

In a group of 20 rabbits, one eye was injected with pegaptanib

(0.3 mg/90 ml; 3.3 mg/ml) immediately after surgery; the other eye

was used as a control and received an injection of 0.9% NaCl (Braun

Medical, Bethlehem, Pennsylvania). For each eye, 90 ml were

injected into the anterior chamber and 90 ml were injected sub-

conjunctivally into the filtration bleb. Rabbits were sacrificed at 3, 8,

14 and 30 days after surgery (n ¼ 5 per time point).

In another group of rabbits (n ¼ 12), repeated injections of

pegaptanib (0.3 mg/90 ml; 3.3 mg/ml) were given every 2 days

subconjunctivally until day 14 and animals were sacrificed 15 and

30 days after surgery (n ¼ 6 per time point).

2.6. Clinical examination and immunohistochemistry

All rabbits were clinically examined and the bleb vascularity

was graded according to the Moorfield bleb grading system

(Wells et al., 2004) on day 1 after surgery and then every 2 days

until the animals were sacrificed. The IOP, the bleb area (width

and length) and height were analyzed under topical anaesthesia

using callipers. Bleb survival was taken as the end-point of the

study; bleb failure was defined as the appearance of a scarred and

flat bleb.

On days 3, 8,14,15 and 30 after surgery, rabbits were killed using

a lethal intravenous injection of sedative. Both eyes were enucle-

ated, fixed overnight in 4% paraformaldehyde (PFA), dehydrated

and embedded in paraffin. Serial sections (7 mm) were immuno-

stained using a murine antibody against human CD31 (M0823;

DakoCytomation A/S, Copenhagen, Denmark) to check the density

of blood vessels and a murine antibody against rabbit CD45

(MC808G; AbD Serotec, Oxford, UK) to analyze inflammation, as

previously described (Li et al., 2009). Deposition of collagen was

analyzed by Sirius Red staining.

Images were obtained using a microscope (Leica Microsystems,

Wetzlar, Germany), equipped with a digital camera (Axiocam

MrC5; Carl Zeiss, Oberkochen, Germany), at a magnification of 20�

and a resolution of 2584 � 1936 pixels. Morphometric analyses

were performed using commercial software (KS300; Zeiss, Ober-

kochen, Germany). The density of blood vessels was determined by

calculating the CD31-positive area as a proportion of the total

wound area. The density of leukocytes was quantified by counting

the CD45-positive cells, expressed in number/mm2 of the total area

of wound healing. Deposition of collagen was determined by

measuring the percentage of collagen fibres in the total wound area

under polarized light.

2.7. Statistical analysis

All in vitro and immunomorphometric data were analyzed using

the Student’s t-test for independent samples. Data at individual

time points were analyzed usingmixedmodel analysis for repeated

measures (using SAS 9.2). KaplaneMeier survival analysis was

performed for bleb failure using the logrank test. P � 0.05 were

considered to be statistically significant. Data are represented as

mean � SEM.

3. Results

3.1. Expression of VEGF-R2 and NRP-1 on HTF and HUVEC

Quantitative real-time RT-PCR on HTF and HUVEC showed

expression of VEGF-R2 and NRP-1 on both cell types. The expres-

sion of VEGF-R2 mRNA in HUVEC was 20 times higher as compared

to HTF (P ¼ 0.02). On the other hand, a 1.3 fold higher expression of

NRP-1 was found on HUVEC as compared to HTF (P¼ 0.04; data not

shown).

3.2. Effect of VEGF isoform administration on proliferation

of HUVEC and TF

To analyze the effect of the VEGF isoforms on proliferation of

HUVEC (as a measure of an angiogenic response) and of HTF and

RTF (as a measure of a fibrotic response), cells were grown in

serum-free medium in the presence of increasing concentrations

of the isoforms. Administration of VEGF121 induced a significant

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699 691

increase in HUVEC proliferation (with 39%, 70% and 60% increase

for 10, 25 and 100 ng/ml, respectively); 25 and 100 ng/ml VEGF121were also able to stimulate both HTF (19% and 18%, respectively)

and RTF (16% and 18%, respectively) (Fig. 1AeC). VEGF165, admin-

istered at 10 and 25 ng/ml, was only able to significantly increase

HUVEC (by 35% and 41%, respectively), but not HTF or RTF prolif-

eration. The highest concentration of VEGF165 (100 ng/ml) signifi-

cantly increased HUVEC as well as RTF growth, respectively by 36%

and 16%. Using this high dose, we also observed a trend towards an

increased HTF proliferation (P ¼ 0.07) (Fig. 1DeF). Conversely,

addition of 25 and 100 ng/ml VEGF189 significantly increased

proliferation of HTF (with 17% and 24%, respectively) and RTF (with

14% and 20%, respectively), whereas no change in HUVEC prolifer-

ation was observed (P > 0.05; Fig. 1GeI).

To exclude an effect of the varyingmolecular weight of the VEGF

isoforms, we repeated the proliferation experiment using 0.25, 1, 2,

5, 10 nM of the different isoforms. The results using molar

concentrations (nM) were comparable to those using weight

concentrations (ng/ml) (data not shown).

Thus, administration of VEGF121 stimulated TF as well as HUVEC

proliferation, whereas VEGF189 only increased the growth of TF

in vitro. A significantly increased proliferation of HUVEC was

noticed after administration of VEGF165. Importantly, the highest

dose of VEGF165 was able to significantly stimulate RTF prolifera-

tion, whereas at this high concentration also amore subtle effect on

HTF growth could be observed.

3.3. Differential activation of the ERK pathway followed by cell

proliferation in HUVEC and HTF

To clarify the differential effects on proliferation elicited by

VEGF121, VEGF165 and VEGF189 (all 25 ng/ml), we compared the

activation of the ERK pathway in HTF and HUVEC. C-Myc activation

was measured as an indication of ERK activation. Spectrophoto-

metric analyses revealed a similar c-Myc activation in non-

stimulated HUVEC and HTF (P ¼ 1.00). Except for VEGF189 on

HUVEC, all isoforms significantly stimulated c-Myc activation

compared to negative control (P < 0.05). As can be appreciated

Fig. 1. HTF, RTF and HUVEC proliferation after VEGF administration (expressed as % of control). All cells were grown in serum-free medium in the presence of VEGF isoforms for

48 h; * ¼ P < 0.05. (AeC) Administration of 25 and 100 ng/ml VEGF121 significantly stimulated the proliferation of HTF, RTF and HUVEC, whereas lower doses did not have any effect.

(DeF) VEGF165 (10 and 25 ng/ml) significantly increased HUVEC proliferation; no effect was detectable on HTF or RTF growth. The highest concentration of VEGF165 (100 ng/ml)

significantly increased HUVEC and RTF growth. A trend towards an increased HTF proliferation was noticed after administration of this high dose (P ¼ 0.07). (GeI) HTF and RTF

proliferation was significantly increased after administration of 25 and 100 ng/ml VEGF189, HUVEC proliferation was not altered by this isoform.

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699692

from Table 1, VEGF121 activated c-Myc (and ERK pathway) in HTF

and HUVEC in a similar way (P ¼ 0.37). In contrast, administration

of VEGF165 resulted in a significantly higher c-Myc activation level

in HUVEC, as compared to HTF (P ¼ 0.02), whereas c-Myc was

significantly more activated in HTF as compared to HUVEC after

VEGF189 administration (P ¼ 0.01; Table 1).

Thus, VEGF121 increased the activation of the ERK pathway in

both HTF and HUVEC, whereas VEGF165 and VEGF189 mainly

induced ERK activation in, respectively HUVEC and HTF, at least as

shown via activation of c-Myc.

3.4. Effect of ERK pathway inhibition (PD98059) on VEGF-induced

cell proliferation

To study the effect of ERK-inhibition on VEGF isoform induced

proliferation, cells were pretreated with PD98059 2 h prior to

VEGF-treatments. Three hours after administration, VEGF121 and

VEGF189 stimulated HTF growth, compared to control (with 3% and

21%, respectively), which was significantly reduced by PD98059

(with 22% and 25%, respectively; data not shown). On the other

hand, HUVEC proliferationwas significantly increased after VEGF121and VEGF165 administration (with 8% and 20%, respectively), which

was significantly inhibited after addition of the ERK-inhibitor (with

21% and 24%, respectively; data not shown).

Thus, VEGF121 and VEGF189 induced HTF proliferation was

significantly reduced by PD98059, whereas the ERK pathway

inhibitor significantly inhibited HUVEC proliferation, induced by

VEGF121 and VEGF165.

3.5. Effect of pegaptanib administration on proliferation

of HUVEC and TF

To evaluate the effect of specific inhibition of VEGF165,

increasing concentrations of pegaptanib (0, 0.03, 0.3, 1 and 2 mg/

ml) were added to HUVEC, HTF and RTF, all growing in complete

medium. A dose-dependent reduction of HUVEC proliferation was

seen 48 h after pegaptanib administration, as compared to

complete medium (P < 0.05 for all concentrations from 0.03 mg/ml

onwards; Fig. 2A). A concentration of 2 mg/ml was necessary to

inhibit the proliferation of HTF (P ¼ 0.009). Comparable to HTF,

2 mg/ml pegaptanib significantly inhibited RTF proliferation

(P ¼ 0.001). Also 1 mg/ml of the aptamer was able to reduce the

growth of RTF (P¼ 0.002), but not of HTF (Fig. 2B, C). In all cases, the

highest dose of pegaptanib (2 mg/ml) inhibited HUVEC, HTF and

RTF to a similar extent as 10 mg/ml bevacizumab (Fig. 2).

Thus, proliferation of HUVEC was significantly inhibited by

pegaptanib in a dose-dependent manner, whereas HTF growth was

only significantly reduced at the highest dose (2 mg/ml). Prolifer-

ation of RTF was reduced already after administration of 1 mg/ml of

the aptamer.

3.6. Effects of pegaptanib in a rabbit trabeculectomy model

To verify the role and relative contribution of VEGF165 in scar

formation after filtration surgery and to determine whether the

VEGF165 selective inhibitor, pegaptanib, is able to improve glau-

coma surgery outcome in vivo, one group of rabbits received one

single injection immediately after surgery. Another group was

treated with repeated injections of pegaptanib every 2 days sub-

conjunctivally until day 14. IOP, bleb area, height and survival were

analyzed as a measure of filtration surgery outcome. A single

injection or repeated injections of pegaptanib did not influence the

IOP; in both groups the pressure was similar in the treated and

control eyes over 30 days (Fig. 4AeE).

The typical appearance of the blebs after a single injection of

pegaptanib on different time points after surgery is shown in

Fig. 3A. One single pegaptanib-injection was associated with an

elevated bleb in the initial days after surgery (bottom panels)

compared to a flat and scarred bleb in the control group (top

panels). A single injection was able to improve the outcome after

surgery by increasing bleb area until day 9 (n ¼ 20; P ¼ 0.0009;

Fig. 4B) and by increasing the bleb height immediately after surgery

(n ¼ 20; P ¼ 0.09; Fig. 4C). No significant differences were seen in

bleb survival (n ¼ 20; P ¼ 0.83; Fig. 4D). (Immuno-)histological

stainings showed a significant reduction of 52% in blood vessel

density in the treated eyes compared to controls, at 3 days after

surgery (n ¼ 5; P ¼ 0.001; Fig. 5A). These results were in keeping

with the Moorfields’ scoring of the blebs during follow-up. After

scoring, a significant reduction in vascularity was seen as well at

postoperative day 3, but not at day 30 (data not shown). Therewere

no significant differences in inflammatory responses (number of

infiltrating leukocytes) and collagen deposition (n ¼ 5; Fig. 5B, C).

In the group with repeated injections, two rabbits died unex-

pectedly during the experiment (one on day 9 and another on day

19 after surgery). It was not clear whether this occurred due to the

treatment or the anaesthesia. In the remaining 10 rabbits, repeated

injections were able to significantly improve the bleb area up until

15 days after surgery (n ¼ 10; P ¼ 0.0001; Fig. 4F). Bleb height was

increased a few days after surgery (n ¼ 10; P ¼ 0.10; Fig. 4G).

Repeated injections significantly prolonged bleb survival after

filtration surgery, as shown in the KaplaneMeier survival curve

(n¼ 10; P¼ 0.05; Fig. 4H). Fig. 3B shows the appearance of the blebs

after repeated pegaptanib-injections (bottom panels) compared to

control eyes (top panels) at different time points. Angiogenesis was

significantly reduced by 43% on day 15 (n ¼ 5; P ¼ 0.001) and by

26% on day 30 (n ¼ 5; P ¼ 0.03) after surgery, as compared to

control conditions (Fig. 5D). Therewere no significant differences in

inflammatory responses and collagen deposition (n ¼ 5; Fig. 5E, F).

Thus, a single administration of pegaptanib at the time of tra-

beculectomy reduced postoperative angiogenesis during the initial

phase, but not inflammation or collagen deposition. Repeated

injections of pegaptanib improved surgery outcome by improving

bleb survival and by reducing angiogenesis up to at least 30 days

after surgery.

4. Discussion

Filtering surgery is the most effective treatment for glaucoma,

and is therefore a crucial procedure in the management of this

blinding disease (Lama and Fechtner, 2003). Unfortunately, exces-

sive postoperative wound healing with subsequent scarring

frequently leads to surgical failure (Addicks et al., 1983). Perioper-

ative anti-mitotics, such as mitomycin-C (MMC) and 5-Fluorouracil

(5-FU) can improve surgical outcome (Katz et al., 1995), but these

antimetabolites carry a risk of vision-threatening complications,

such as scleral thinning and infections (Gillies and Su, 1991;

Greenfield et al., 1998; Hitchings and Grierson, 1983; Jampel

et al., 1988; Katz et al., 1995; Lama and Fechtner, 2003; Skuta and

Parrish, 1987). Furthermore, blocking TGF-b, which seemed prom-

ising in animal models (Cordeiro et al., 2003), was not efficient in

Table 1

Activation of c-Myc in HTF and HUVEC, as an indication of ERK activation. Data are

represented as mean � SEM.

C-Myc activation

(OD at 450 nm)

HTF HUVEC P-value

(HTF vs HUVEC)

0.1% FBS 0.32 � 0.04 0.32 � 0.01 1.00

VEGF121 0.44 � 0.03 0.51 � 0.08 0.37

VEGF165 0.44 � 0.03 0.67 � 0.11 0.02

VEGF189 0.47 � 0.03 0.30 � 0.01 0.01

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699 693

a clinical study (Khaw et al., 2007). Therefore, there is still a need

for alternative strategies to prevent filtration failure.

Wound healing occurs through a combination of four processes:

clot formation, angiogenesis, inflammation and collagen deposition

(Addicks et al., 1983; Gillies and Su, 1991; Lama and Fechtner, 2003;

Lee et al., 1995). VEGF is a well-known key player in angiogenesis

(Carmeliet et al., 1996; Dvorak et al., 1999; Kowanetz and Ferrara,

2006). An additional role in inflammation and fibrosis has been

suggested, but has been studied much less extensively (Beddy et al.,

2004;Murakami et al., 2006;Wilgus et al., 2008). AsVEGFmight play

a role in several processes of wound healing (Li and Li, 2003; Nissen

et al., 1998; Wilgus et al., 2008), we hypothesized that this growth

factor might be a good target candidate for anti-fibrotic adjunctive

strategies in glaucoma surgery. Indeed, we recently published

encouraging in vitro and in vivo data indicating that VEGF as well as

its receptors are expressed by Tenon fibroblasts. Moreover, we

showed that non-selective VEGF inhibition by bevacizumab blocks

fibroblast proliferation in vitro, aswell as postoperative angiogenesis

and fibrosis after filtering surgery in vivo (Li et al., 2009). In addition,

Memerzadeh et al. showed that repeated injections of bevacizumab

were able to reduce both vascularity andfibrosis in a rabbitmodel for

trabeculectomy (Memarzadeh et al., 2009). These experiments were

performed with the non-isoform selective anti-VEGF antibody bev-

acizumab. However, other anti-VEGF treatments are used in the

ophthalmological practice, some of which have more selective

properties. Importantly, we previously reported that various VEGF

isoforms exert a different function during physiological angiogenesis

(Stalmans et al., 2003, 2002). Their differential role in pathological

angiogenesis has also been studied, but unfortunately still remains

rather controversial (Ishida et al., 2003b; Usui et al., 2004).

Furthermore, the specific role of the VEGF isoforms in inflammation

(Ishida et al., 2003a) and fibrosis (Roberts et al., 2007) remains

largely unknown.

Using in vitro experiments, the current study revealed that

VEGF121 and VEGF165 significantly stimulate proliferation of

HUVEC, by activating the ERK pathway. Moreover, PD98059, an

inhibitor of the ERK pathway, significantly inhibited VEGF121 and

VEGF165-induced HUVEC proliferation. These data are in keeping

with the observations by Zhang et al. (Zhang et al., 2008), who

recently reported that VEGF165, and to a lesser extent VEGF121,

stimulated HUVEC proliferation. These authors showed that

VEGF165 significantly increased the level of phosphorylation in the

MEK/ERK pathway, whereas VEGF121 had little effect. For the first

time, we report here the effect of the different isoforms on TF

growth. We showed that TF proliferation was mainly induced by

VEGF121 and VEGF189, associated with an activation of the ERK

pathway. These proliferative effects were significantly reduced by

the ERK pathway inhibitor PD98059. Also at the highest concen-

tration, VEGF165 was able to significantly increase RTF prolifera-

tion, whereas a subtle effect of high VEGF165 levels on HTF could

not be excluded. Our results indicate that VEGF165 is the most

important isoform in inducing HUVEC proliferation, while VEGF189is the most prominent in inducing TF proliferation. VEGF121 clearly

affects both cell types. Additionally, we demonstrated that HUVEC

were effectively inhibited by pegaptanib (a selective VEGF165-

inhibitor) in a dose-dependent way. HTF growth was only signif-

icantly reduced using the highest dose of this selective inhibitor.

Proliferation of RTF was already reduced after administration of

lower doses of the aptamer. Our findings thus indicate that HTF

might be somewhat less sensitive compared to RTF as more

VEGF165/pegaptanib is required for a significant HTF stimulation/

inhibition.

Fig. 2. HUVEC, HTF and RTF proliferation after pegaptanib administration (expressed as % of control). All cells were grown in complete medium in the presence of pegaptanib for

48 h; * ¼ P < 0.05. (A) Pegaptanib significantly inhibited the proliferation of HUVEC in a dose-dependent manner, at a minimal dose of 0.03 mg/ml. (B) A concentration of 2 mg/ml

was necessary to inhibit the proliferation of HTF (P ¼ 0.009). (C) Comparable to HTF, 2 mg/ml pegaptanib inhibited RTF proliferation significantly (P ¼ 0.001). Furthermore, even

1 mg/ml of the aptamer was able to reduce the growth of RTF (P ¼ 0.002). In all cases, the highest dose of pegaptanib (2 mg/ml) inhibited HUVEC, HTF and RTF to a similar extent as

10 mg/ml bevacizumab.

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699694

The functional difference of the various isoforms on HTF and

HUVEC proliferation can be attributed to different expression

levels of VEGF-R2 and/or VEGF-R co-receptors on HTF and

HUVEC. Both HTF (Li et al., 2009) and HUVEC (Castro-Rivera et al.,

2004) are known to express VEGF-R2 but, of note, a lower

expression of this receptor on synovial fibroblasts as compared to

HUVEC has been reported (Kim et al., 2006). Our real-time RT-

PCR analyses indeed revealed that the expression of VEGF-R2

mRNA on HUVEC was 20 times higher as compared to HTF.

Additionally, HUVEC (Castro-Rivera et al., 2004; Deroanne et al.,

2002; Kokolakis et al., 2006; Soker et al., 1998; Tirand et al.,

2006) and certain types of fibroblasts (Haag et al., 2010; Kim

et al., 2006) were also reported to express NRP-1, but whether

NRP-1 is expressed on Tenon fibroblasts remained unknown. Our

RT-PCR data showed that HTF indeed express NRP-1, but a lower

level as compared to HUVEC.

Our expression data may explain the difference in activation of

both cell types by the various VEGF isoforms. Indeed, VEGF121,

lacking the VEGF-R co-receptor binding domains (Robinson and

Stringer, 2001), exerts proliferative effects on both cell types by

binding VEGF-R2. However, similar concentrations of VEGF121 seem

to stimulate HUVEC more than TF, probably because of the lower

expression of VEGF-R2 on HTF. VEGF165 binds VEGF-R2 but also

strongly binds NRP-1. As such, the reduced expression of VEGF-R2,

and possibly of NRP-1, on HTF may explain why VEGF165 seems to

be more potent in inducing proliferation of HUVEC as compared to

HTF. In comparison to VEGF121 and VEGF165, VEGF189 strongly binds

HSPGs and is believed to constitute a reservoir of growth factor,

which can be slowly released by exposure to urokinase plasmin-

ogen activator (uPA) (Robinson and Stringer, 2001). Indeed, protein

folding of this larger isoform obscures regions responsible for

receptor binding and therefore native VEGF189 is unable to bind

VEGF-R2 and NRP-1 (Plouet et al., 1997). Cleavage of VEGF189 by uPA

is required for its receptor binding and biological actions. Impor-

tantly, zymographic analyses did reveal the presence of uPA in cell

lysates of conjunctival fibroblasts (Chao et al., 2011), but not of

HUVEC (Herve et al., 2005). These findings may explain our

observation that VEGF189 could stimulate HTF, but not HUVEC,

however, further research is required. Moreover, we cannot exclude

that the observed VEGF isoform specific effects on the two cell

types may also be explained by a direct or indirect activation of

different signal transduction pathways upon binding VEGF-R2.

Based on the in vitro results we hypothesized that, whereas we

previously published that non-selective VEGF inhibition is anti-

angiogenic as well as anti-fibrotic (Li et al., 2009), selective

VEGF165 blockage might mainly affect angiogenesis. Our in vivo

surgery experiments indeed confirmed that a single administration

of pegaptanib significantly impaired angiogenesis, but did not

reduce collagen deposition. Of note, neither selective nor non-

selective VEGF inhibition could reduce the amount of post-

operative inflammatory responses inour trabeculectomymodel (this

study and Li et al., 2009). Besides a difference in isoform selectivity,

the greater potency of bevacizumab compared to pegaptanib to

inhibit postoperative scarring after a single peroperative adminis-

trationmight be attributed to a difference in time of action between

both inhibitors. Indeed, the half-life of intra-ocular bevacizumab

Fig. 3. Macroscopic postoperative photographs of eyes after surgery. Arrows: edges of the blebs. (A) Macroscopic photographs of eyes after a single pegaptanib-injection show

surviving blebs that remain diffusely elevated (bottom) compared to control (top) until day 8. At day 14 the blebs were comparable. (B) Repeated injections of pegaptanib were

associated with an elevated bleb until day 15 after surgery (bottom) compared to a flat and scarred bleb in the control group (top).

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699 695

(4.88 days) (Bakri et al., 2007) was shown to be significantly longer

than that of pegaptanib (3.91 days) (Apte et al., 2007). To further

elucidate whether the observed difference in anti-fibrotic efficacy

was caused by a difference in half-life, we extended the treatment by

administering additional repeated postoperative injections of

pegaptanib after the initial peroperative treatment. Although the

effect on postoperative outcome slightly improved, this effect was

still solely attributable to the anti-angiogenic properties of the

aptamer and not to its effects in collagen deposition. In both groups

(single and repeated injections of pegaptanib) no significant changes

in IOP compared to placebo were observed. However, this is in

keeping with the previous literature describing this model for sub-

conjunctival scarring (Cordeiro et al., 2003; Mead et al., 2003;

Memarzadeh et al., 2009), and can be explained by the fact that

the surgery is performed on non-glaucomatous rabbit eyes with

normal IOPs at baseline.

In summary, our in vitro and in vivo data indicate that selective

VEGF165 inhibition by pegaptanib is less effective than non-

selective VEGF inhibition by bevacizumab (Li et al., 2009) in

reducing scarring after glaucoma surgery, presumably due to

retained action of VEGF121 and VEGF189 isoforms, which have

a more pronounced effect on the Tenon fibroblasts as compared to

Fig. 4. Clinical investigation of eyes after surgery and pegaptanib administration. (A) A single injection or (E) repeated injections of pegaptanib did not influence the IOP; in both

groups the pressure was similar in the treated and the control eyes over 30 days. (B) A single injection of pegaptanib significantly improved the outcome of the surgery by increasing

the bleb area until day 9 (P ¼ 0.0009) and (C) by increasing the bleb height immediately after surgery (P ¼ 0.09). (D) No significant differences were seen in bleb survival (P ¼ 0.83).

(F) Repeated pegaptanib-injections significantly improved the bleb area up until 15 days after surgery but not thereafter (P ¼ 0.0001). (G) Bleb height was increased a few days after

surgery (P ¼ 0.10). (H) As shown in the KaplaneMeier survival curve, repeated injections significantly prolonged bleb survival after filtration surgery, compared to control (P ¼ 0.05).

T. Van Bergen et al. / Experimental Eye Research 93 (2011) 689e699696

VEGF165. Although proliferation assays may not necessarily trans-

late to clinical relevance, these data suggest that the VEGF isoforms

play distinct roles in angiogenesis and wound healing, and that

non-selective VEGF inhibition has a greater anti-scarring potency

than selective VEGF165 blockage. Our study sheds new light on the

differential function of the VEGF isoforms in the processes of

angiogenesis and scar formation. These insights may have impor-

tant therapeutic implications not only for glaucoma surgery, but

also for modulation of postoperative scarring in general.

5. Conclusions

Our data indicate that VEGF isoforms play a differential role not

only in ocular angiogenesis but also in wound healing. VEGF165 and

VEGF121 predominantly affect blood vessel growth, whereas

VEGF189 may be more important in fibrosis. Selective inhibition of

VEGF165 may therefore be less effective to reduce ocular scar

formation than non-selective VEGF inhibition. These findings

reveal new insights in the role of the different isoforms in the

process of wound healing.

Disclosure statements

Tine Van Bergen: none; Evelien Vandewalle: none; Sara Van de

Veire: none; Mieke Dewerchin: none; Jean-Marie Stassen: none;

Lieve Moons: none; Ingeborg Stalmans: none.

Conflict of interest

Pegaptanib was kindly provided by Pfizer.

Acknowledgements

The authors wish to thank Sofie Beckers, Magda Bressinck and

Joris Souffreau for their technical support; and Silvia Cecere for

statistical advice.

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