antifibrotic activity of bevacizumab on human tenon's fibroblasts in vitro

Antifibrotic Activity of Bevacizumab on Human Tenon’sFibroblasts In Vitro

Evelyn C. O’Neill,1,2 Queena Qin,1,2 Nicole J. Van Bergen,1 Paul P. Connell,1

Sushil Vasudevan,1,3 Michael A. Coote,1 Ian A. Trounce,1 Tina T. L. Wong,4

and Jonathan G. Crowston1

PURPOSE. To evaluate the effect of the anti–VEGF-A monoclonalantibody bevacizumab on primary human Tenon’s capsulefibroblasts (HTFs) in an in vitro model of wound healing.

METHODS. Fibroblasts were cultured in RPMI media, andbevacizumab was administered at a concentration rangingfrom 0.25 to 12.5 mg/mL. Fibroblast viability and cell deathwere assessed using the MTT colorimetric assay, lactatedehydrogenase assay, BrdU assay, and live/dead assay. Fibro-blast contractility was assessed in floating collagen gels.Morphologic changes were assessed by transmission elec-tron microscopy. Antifibrosis activities were compared with5-fluorouracil.

RESULTS. Bevacizumab induced a significant dose-related reduc-tion of HTF cell number at 12.5 mg/mL at 72 hours (P ! 0.05).Under serum-free conditions, bevacizumab induced significantfibroblast cell death at concentrations greater than 7.5 mg/mL(P ! 0.05). Bevacizumab caused a moderate inhibition offibroblast gel contraction from baseline (P ! 0.05). Scanningelectron microscopy revealed marked vacuolization in bevaci-zumab-treated fibroblasts.

CONCLUSIONS. Bevacizumab disrupted fibroblast proliferation,inhibited collagen gel contraction ability, and induced fibro-blast cell death at concentrations greater than 7.5 mg/mL inserum-free conditions. These results demonstrated that bevaci-zumab inhibited a number of fibrosis activities in culture.These activities may underpin the antifibrosis effect proposedin vivo. (Invest Ophthalmol Vis Sci. 2010;51:6524–6532) DOI:10.1167/iovs.10-5669

Excess scarring at the site of a filtering bleb is the mostcommon cause of failure after glaucoma filtration sur-

gery.1–3 Tenon’s fibroblasts are the main effector cells in theinitiation and mediation of wound healing and fibrotic scarformation after trabeculectomy.3 Success rates of filtrationsurgery have significantly improved with the use of adjunc-tive antifibrotic agents such as 5-fluorouracil (5-FU) andmitomycin C (MMC).4 – 6 These agents, however, can induce

significant cell death in treated tissues7 that may contributeto antifibrosis activity but simultaneously predispose to po-tentially sight-threatening complications, including hypot-ony, wound leak, blebitis, and endophthalmitis.8 Despitethese treatments, a number of patients continue to scarexcessively, and improved methods for titrating and control-ling the wound healing response are sought.

Angiogenesis, the formation of new capillaries from pre-existing blood vessels, occurs in both health and disease. Itis implicated in tumorigenesis and metastasis,9 rheumatoidarthritis,10 and blinding ocular conditions including prolif-erative diabetic retinopathy11 and choroidal neovasculariza-tion in age-related macular degeneration (ARMD).12 Angio-genesis is also a critical component of wound healingbecause it allows early migration of inflammatory cells andfibroblasts into the wound. The family of vascular endothe-lial growth factors (VEGF) has been identified as the primaryregulators of angiogenesis13–15 and VEGF-A as the primaryregulator driving angiogenesis. As a result multiple antian-giogenic agents, targeting VEGF-A or its receptor VEGFR2,have been developed to specifically target and treat VEGF-A– driven ocular pathology.14

Anti-VEGF monoclonal antibodies have been developedto treat solid tumors16 –18 and now also form part of theclinical management of ocular neovascular diseases.19 Bev-acizumab (Avastin; Genetech, Inc., South San Francisco, CA)is a full-length humanized monoclonal antibody directedagainst all isoforms of VEGF-A (VEGF-121, -145, -165, -183,-189, and -209) and is approved by the US Food and DrugAdministration for the treatment of metastatic colorectal andmetastatic breast cancer.17,18 It binds and neutralizes allhuman VEGF-A isoforms and bioactive proteolytic frag-ments.20 Wound healing complications, including early de-layed wound closure or dehiscence in anastomosis (aftercolorectal surgery), have been reported after the adminis-tration of intravenous bevacizumab.21,22 This dehiscencemay occur several months after the original surgical anasto-moses, suggesting bevacizumab may induce long-term inhi-bition of wound healing.

Intravitreal use of bevacizumab has a good safety profilein humans,23 and there are several reports of its use inproliferative diabetic retinopathy, ARMD, inflammatory oc-ular neovascularization, and neovascular glaucoma.24 –30

There are also isolated case reports of its use in glaucomafiltration surgery, particularly in neovascular glaucoma.31,32

However, to date, the efficacy of bevacizumab in filtrationsurgery, specifically the effect of bevacizumab on humanTenon’s fibroblasts (HTFs) in culture, is largely unknown.

In this study, we describe the effects of the anti–VEGF-Amonoclonal antibody bevacizumab on HTF in an in vitro modelof wound healing.

From the 1Centre for Eye Research Australia, the Royal VictorianEye and Ear Hospital, East Melbourne, Victoria, Australia; 3Faculty ofMedicine, Universiti Teknologi MARA, Shah Alam, Malaysia; and 4Sin-gapore National Eye Center and Singapore Eye Research Institute,Singapore, Republic of Singapore.

2These authors contributed equally to the work presented hereand should therefore be regarded as equivalent first authors.

Submitted for publication April 9, 2010; accepted May 25, 2010.Disclosure: E.C. O’Neill, None; Q. Qin, None; N.J. Van Bergen,

None; P.P. Connell, None; S. Vasudevan, None; M.A. Coote, None;I.A. Trounce, None; T.T.L. Wong, None; J.G. Crowston, None

Corresponding author: Evelyn C. O’Neill, Centre for Eye ResearchAustralia, the Royal Victorian Eye and Ear Hospital, 32 Gisborne Street,East Melbourne, VIC 3002, Australia; [email protected].

Glaucoma

Investigative Ophthalmology & Visual Science, December 2010, Vol. 51, No. 126524 Copyright © Association for Research in Vision and Ophthalmology

Porcelet Que

Porcelet Que

METHODS

Human Tenon’s Fibroblast Explant CultureHTFs were propagated from explanted subconjunctival Tenon’s capsuleisolated during glaucoma filtration surgery, as described previously.33 Thetenets of the Declaration of Helsinki were observed, institutional humanethics committee approval was granted, and written informed consentwas obtained from all patients. Explanted tissue was attached to thebottom of a six-well plate (Greiner Bio-One, Jena, Germany) with a sterilecoverslip and overlaid with RPMI (Sigma-Aldrich, St. Louis, MO). Allculture media were supplemented with L-glutamine 2 mM, penicillin100,000 U/L, and streptomycin 10 mL/L (all Sigma-Aldrich). For propaga-tion of fibroblasts, the media were also supplemented with fetal calf serum(FCS; 10% of final volume; JRH Biosciences, Lenexa, KS). HTFs wereroutinely cultured in RPMI media, as described. Once the monolayersreached confluence, fibroblasts were passaged and subcultured in 175-cm2 tissue culture flasks (Greiner Bio-One). Cells were incubated at37°C/5% CO2 in a humidified incubator.

Fibroblast Proliferation and Viability StudiesThe MTT assay is a convenient and accurate way of determiningmammalian cell viability. This colorimetric assay quantifies the

number of metabolically active cells based on the cleavage of theyellow tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetrazolium bromide) to purple formazan crystals. The assay wasperformed according to the manufacturer’s instructions (Cell Pro-liferation Kit 1; Roche Diagnostics, Basel, Switzerland). Fibroblastswere plated at 5000 cells/well in 96-well plates and incubated, onattachment, in 50 !L media with bevacizumab (Pharmatel FreseniusKabi, Bad Homborg, Germany) diluted to concentrations of 0.25,2.5, 5, 7.5, 10, and 12.5 mg/mL for 24, 48, and 72 hours. Experi-ments were performed in both 10% FCS media and serum-free mediaconditions. Viability experiments were repeated comparing bevaci-zumab with two isotype control antibodies: a humanized monoclo-nal IgG1" chimeric antibody in which variable regions are deimmu-nized and directed toward fibrin degradation products (proprietaryof and kindly supplied by Agen Ltd., Melbourne, Australia) and anonhumanized IgG isotype.

Cell death was quantified using the lactate dehydrogenase (LDH)assay, based on the measurement of LDH activity released from thecytosol of dying cells into the supernatant. Fibroblasts were plated at5000 cells/well in 96-well plates and were treated with increasingconcentrations of bevacizumab to 12.5 mg/mL, as described. Experi-ments were performed in serum-free media conditions because of

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FIGURE 1. Bevacizumab-treated HTFs(continuous 72-hour) MTT assay. (A)10% FCS media conditions. Reduction inMTT absorbance at 12.5 mg/mL bevaci-zumab in cells in media with 10% FCS(P ! 0.05). *P ! 0.05 with respect tocells in media with 10% FCS with nobevacizumab treatment. (B) Serum-freeconditions MTT assay. Reduction inMTT absorbance at concentrations of 5mg/mL to 12.5 mg/mL in cells in media(P ! 0.05). Significant increase in absor-bance at a concentration of 2.5 mg/mLbevacizumab compared with controlcells (P ! 0.05; n " 3). *P ! 0.05 withrespect to cells in serum-free media withno bevacizumab treatment (control).

IOVS, December 2010, Vol. 51, No. 12 Antifibrotic Activity of Bevacizumab 6525

intrinsic LDH activity in serum. LDH was measured only at the 72-hourtime point. At 72 hours, 80 !L supernatant (media) was transferred toa new 96-well plate, taking care not to disturb the monolayer offibroblasts. The assay was performed according to the manufacturer’sinstructions (Cytotoxicity Detection Kit; Roche Diagnostics), and 1%Triton X-100–treated cells served as positive controls for 100% celldeath.

Fibroblast proliferation was measured with a 5-bromo-2-de-oxyuridine (BrdU) assay that quantitates BrdU uptake into newlysynthesized DNA of replicating cells. HTFs (1 # 104) were platedinto separate wells of a 24-well plate and incubated with BrdU afterbevacizumab treatment for 24 and 48 hours. Proliferating cells weredetected by BrdU labeling directed by the BrdU detection kit man-ufacturer’s protocol (Kit I; Roche Diagnostics). Five hundred to1000 cells were randomly counted in each well. The average ofthree wells per sample was counted. The BrdU labeling index wascalculated as the percentage of BrdU-positive cells. Representativeimages were captured using an epifluorescent inverted phase-con-trast microscope (TE2000S; Nikon, Tokyo, Japan) at 100# magnifi-cation.

Viability/cytotoxicity assay (Live/Dead; Invitrogen MolecularProbes, Carlsbad, CA) was used to determine the ratio of live and deadfibroblasts after bevacizumab treatment. Dye concentrations for cal-cein AM and ethidium homodimer-1 (EthD-1) were optimized accord-ing to the kit’s protocol before experimentation. HTFs were incubated

in serum-containing and serum-free RPMI. HTFs were treated withbevacizumab (10 mg/mL) in accordance with the previous protocol.Control wells were composed of 1% digitonin (for 100% cell death) and50% PBS mixture with culture media; 48-well plates were then incu-bated at 24, 48, and 72 hours. At each time point, the correspondingwells were washed with 1 mL PBS to remove any esterase activitypresent in serum-supplemented RPMI because serum esterase canhydrolyze calcein AM and cause increased extracellular fluorescence.The fibroblasts were then incubated with the dyes calcein AM andEthD-1 at room temperature for 30 minutes. Cells were washed withPBS and viewed under a fluorescence microscope. Five random fieldsfrom each well were photographed and counted.

Floating Collagen Contraction Studies

To assess the influence of bevacizumab on fibroblast contraction,we measured the contraction of fibroblast-seeded collagen gels.First, fibroblasts were resuspended at a density of 5 # 105 cells/mLin either 10% FCS (JBH Biosciences, Lenexa, KS) or concentratedserum-free medium. Each gel was made from 125 !L of 4# concen-trated medium and 200 !L dialyzed collagen 0.1% acetic acid andmixed gently. Sodium hydroxide (0.1 M) was then added to the gelto return the solution to physiological pH and to precipitate thecollagen. Thirty microliters of fibroblasts (at 5 # 105 cells/mL) werethen seeded into the neutralized gel and resuspended briefly. This

FIGURE 2. Bevacizumab inhibition ofHTF proliferation as measured withBrdU assay. HTFs were (A) untreatedcells and (B) cells incubated in bevaci-zumab (10 mg/mL). Nonproliferatingcells stained blue, and proliferatingcells stained green. (C) Histogram ofBrdU positivity in bevacizumab-treatedcells. Values plotted are mean $ SD(n " 3). *P ! 0.05 with respect tountreated cells.

6526 O’Neill et al. IOVS, December 2010, Vol. 51, No. 12

was added to a well of a 48-well plate and incubated at 37°C for 15minutes to set. Three hundred microliters of bevacizumab (PFK 25mg/mL) diluted in RPMI (10% FCS, L-glutamate, penicillin/strepto-mycin) to concentrations of 2.5, 7.5, and 12.5 mg/mL was added tothe wells containing solidified collagen gels. The gels were thengently detached from the wells, and culture medium was added.The free-floating fibroblast-populated collagen gels were incubatedfor 7 days. Images of collagen gels were taken with a digital camera(CyberShot DSC-S700; Sony, Tokyo, Japan) on days 0, 1, 2, 3, and 7.The images were then measured and assessed using ImageJ software(developed by Wayne Rasband, National Institutes of Health, Be-thesda, MD; available at http://rsb.info.nih.gov/ij/index.html).

Electron MicroscopyCells treated with bevacizumab (10 mg/mL), and controls in serumand in serum-free RPMI were prepared as follows. Suspended HTFswere fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer and 2%

osmium tetroxide in distilled water for 30 minutes, respectively,and spun at 3000 rpm for 4 minutes to obtain a single-cell pellet.The pellet was then washed 2 # 10 minutes with distilled water andspun down to reach a concentrated pellet. Then 1% agar waspoured over the pellet and was allowed to solidify at 4°C, afterwhich it was sliced into pieces of 1 mm3 and processed in 3% uranylacetate, followed by dehydration with acetone for routine electronmicroscopy. Each piece of pellet was embedded in an individualmold, and the resin was cured in a 60° oven for 24 hours. Specimensections 50- to 90-!m thick were cut using a microtome (Ultracut;Reichert-Jung, Wetzlar, Germany) and a diamond knife (Diatome,Biel, Switzerland), collected on 200-mesh copper grids, and post-stained in 5% uranyl acetate in ethanol (10 minutes) and Reynold’slead citrate (5 minutes). Specimens were viewed with a transmis-sion electron microscope (CM10; Philips, Eindhoven, The Nether-lands) operating at 60,000 V. Negatives were converted to digitalimages (Photoshop 7; Adobe, Mountain View, CA).

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Statistical AnalysisWhere practical, each laboratory experiment was performed with atleast three replicates per treatment group. Data were assumed to

follow the normal distribution. Statistically significant differencesbetween two data points were analyzed using the paired t-test.Statistically significant differences among three or more data points

FIGURE 4. Effect of bevacizumab onHTF cell death. Live/dead assay wasused to score the number of live(green) to dead (red) cells at 24 and 72hours after treatment. (A) HTFs wereincubated in RPMI $ bevacizumab incycling cells compared with digitonincontrol. (B) HTF in serum-free me-dia $ bevacizumab in noncycling cellscompared with digitonin control. (C)Histogram of cell death in bevaci-zumab-treated cells. Bevacizumab (10mg/mL) induced significant fibroblastcell death at 72 hours in serum-freeconditions (n " 3).


were analyzed using the repeated-measures one-way ANOVA, fol-lowed by the Bonferroni post test in some cases. Data were deemedstatistically significant at P ! 0.05. All analysis was performed usingMicrosoft software packages (Excel; Microsoft, Redmond, WA).

RESULTS

Proliferation Inhibition of HTFs by Bevacizumab

The effect of bevacizumab on fibroblast proliferation wasquantified with MTT assay to determine the number ofviable cells. A statistically significant decrease in fibroblastnumber was observed at a concentration of bevacizumab of12.5 mg/mL (P ! 0.05) in cells with 10% FCS (Fig. 1A). Inserum-free conditions, there was a significant reduction infibroblast number at a concentration of bevacizumab of 5.0mg/mL to 12.5 mg/mL (P ! 0.05; Fig. 1B). Interestingly,there was a statistically significant increase in fibroblasts ata concentration of 2.5 mg/mL bevacizumab in serum-freeconditions. (P ! 0.05). Proliferation rates were also mea-sured using BrdU incorporation. This demonstrated signifi-cant inhibition at 48 hours with bevacizumab (10 mg/mL)compared with controls (P ! 0.05; Fig. 2).

Bevacizumab-Induced Fibroblast Cell Death

The LDH assay was then used to assess cumulative cell death72 hours after bevacizumab treatment. In serum-free condi-tions, bevacizumab at 7.5 mg/mL or greater had a significantcytotoxic effect on HTFs (P ! 0.001, paired t-test). Treatmenteffect appeared to plateau beyond 7.5 mg/mL (Fig. 3A). Thiswas correlated with a reduction in cell numbers determined byMTT assay beyond 7.5 mg/mL bevacizumab (Fig. 3B).

We next investigated the effect of serum on bevacizumab-induced cell death using a live/dead assay in bevacizumab (10mg/mL), and digitonin-treated cells (positive controls) in se-rum-containing and serum-free conditions given that LDH as-says cannot be performed in serum-containing conditions be-cause of the intrinsic activity of LDH in serum. At 10 mg/mL,bevacizumab induced no significant cell death in 10% FCS to 72hours (Fig. 4A). However, it induced almost 100% fibroblastcell death at 24 hours in serum-free conditions. (Figs. 4B, 4C).

To confirm that the effect on fibroblast viability was specificto bevacizumab and not a nonspecific consequence of highantibody concentration, we compared proliferation assays of

bevacizumab with two isotype control antibodies. Experi-ments were performed in serum-free conditions comparing theeffect of 7.5 mg/mL bevacizumab (known to inhibit prolifera-tion in serum-free conditions) with 7.5 mg/mL isotype controlantibodies. The humanized control antibody had no significanteffect on the number of viable cells. There was a statisticallysignificant decrease in the number of viable fibroblasts withbevacizumab-treated cells only (P ! 0.05; Fig. 5).

Bevacizumab Effect on HTF-Mediated CollagenGel Contraction

Bevacizumab at 12.5 mg/mL had a moderate inhibitory effecton collagen gel contraction compared with untreated cells(P ! 0.001, two-way ANOVA; Fig. 6A). A dose response wasseen between 2.5 mg/mL and 12.5 mg/mL bevacizumab treat-ment (Fig. 6B). Under serum-free conditions, 12.5 mg/mL be-vacizumab increased the inhibition of collagen gel contraction(78% collagen gel area) compared with controls (27% collagengel area; Fig. 6C). Inhibition of HTF contraction with bevaci-zumab was increased in serum-free conditions.

Morphologic Changes in HTF after BevacizumabTreatment: Phase-Contrast Microscopyand Transmission Electron Microscopy

Analysis of cell morphology under phase-contrast (Fig. 7) andthe live/dead assay (Figs. 4A–C) showed that under serum-freeconditions, 10 mg/mL bevacizumab induced high levels of celldeath. To assess the mode of cell death, scanning electronmicroscopy examining the nuclear morphology of HTF afterbevacizumab (10 mg/mL) treatment was performed (Fig. 8). Inbevacizumab-treated fibroblasts, there was the characteristicmorphologic appearance seen in serum-free cells. Transmis-sion electron microscopy images revealed significant vacuol-ization of internal HTF cell cytoplasm with complete lysis ofHTF cell membranes and cell debris.

DISCUSSION

In the present study, we demonstrated that the anti–VEGF-Amonoclonal antibody bevacizumab induced potent antifibroticactivity through significant reduction in fibroblast viability,inhibition of HTF proliferation, induction of low levels of cell

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death, and inhibition of cell-mediated collagen gel contractionin vitro. The concentrations of bevacizumab required to inducecell death were higher in serum-containing conditions than inserum-free conditions because VEGF in serum binds bevaci-zumab and inactivates it.

Excessive postoperative scarring is the most common causeof failed glaucoma filtration surgery. Intraoperative MMC and5-FU are clinically used to inhibit fibrosis and improve surgicaloutcomes.4–6 However, despite their use, a significant failurerate persists, with an associated increase in other postoperativecomplications. VEGF is a key molecule in the wound healingresponse.34 Humanized anti–VEGF-A monoclonal antibodies(bevacizumab and ranibizumab) are used widely in ophthal-mology to inhibit angiogenesis in ARMD and to enable a goodsafety profile with ocular administration.25–28

Angiogenesis plays a vital role in wound repair, facilitatingwound closure by enabling inflammatory cells and fibroblasts

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FIGURE 6. Floating collagen gel contraction studies. (A) Floatingcollagen gel contraction studies, 7 days (n " 3; P ! 0.05, two-wayANOVA). (B) Floating collagen gel contraction studies seeded withHTF prepared in 100% serum and treated with bevacizumab con-centrations of 2.5 mg/mL, 7.5 mg/mL, and 12.5 mg/mL (n " 3). (c)Floating collagen gel contraction studies, 7 days (HTFs resuspendedin serum-free medium), bevacizumab 12.5 mg/mL (n " 3; P !0.001, two-way ANOVA).

FIGURE 7. Effect of serum and bevacizumab on HTF morphology (n " 3).

FIGURE 8. Scanning electron microscopy images of HTFs. (A) HTFsincubated in RPMI with 10% serum showing normal cellular structureand normal nucleus and cytoplasm. (B) HTFs incubated in serum-freeRPMI. (C) HTFs incubated in bevacizumab 10 mg/mL with 10% serumshowing significant vacuolization of cytoplasm (arrows). (D) HTFsincubated in serum-free bevacizumab 10 mg/mL showing only cellulardebris.


to migrate to the wound and by providing the vascular scaffoldfor granulation tissue formation.34 Vascular remodeling occursbecause of the carefully balanced interplay of proangiogenicand antiangiogenic factors. Both angiogenic agonists and an-tagonists have been identified at various stages of wound re-pair. It is now widely established that VEGF-A is responsible fornormal vasculogenesis, hemangiogenesis, and lymphangiogen-esis.13,35–37 Despite this, however, relatively little attention hasbeen given to the reported antifibrotic effects of VEGF-A.

In addition to the expected elevated levels of VEGF-A in ocularfluids, diabetic retinopathy, and other retinal vascular disorders,11

raised VEGF-A levels in the aqueous humor of patients withnonneovascular glaucoma have also been reported.38,39 This in-crease in VEGF in the aqueous humor of glaucoma patients maycontribute to postoperative inflammation and fibrosis. VEGF-Areceptors have also recently been shown to be expressed inHTF.39 Thus, targeting the VEGF-A molecule would appear to bea plausible method of reducing the post-operative scarring re-sponse after glaucoma filtration surgery.

VEGF inhibition has also been shown to attenuate fibrosis ina murine model of allergic airway disease,40 to induce a profi-brogenic gene expression profile in glomerular endothelial celllines,41 and to reduce fibrosis in cutaneous wounds of adults.42

Recent animal studies using the rabbit model of trabeculec-tomy, a model with a known vigorous wound healing re-sponse, have shown subconjunctival bevacizumab significantlyimproved glaucoma filtration surgery success and bleb surviv-al39,43 because of the combined inhibition of angiogenesisduring the initial phase of healing and the reduced fibrosis atlater stages of wound repair. Li et al.39 found a single dose of0.75 mg bevacizumab (0.3 mL of 25 mg/mL) given immediatelyafter surgery significantly reduced the density of blood vesselsduring the early stages of wound healing and reduced collagendeposition in the later stages. Memerzadeh et al.,43 using seveninjections of 1.25 mg bevacizumab (0.05 mL of 25 mg/mL)during the first 14 days after surgery found it reduced collagenand elastic fiber deposition, reduced fibroblast differentiationinto myofibroblasts, and resulted in a loss of fibroblast mitoticactivity. Recent clinical reports have explored the use of bev-acizumab after glaucoma filtering surgery, indicating that theagent can be administered safely at the time of surgery or in thepostoperative period.32,44–46 In the largest of these, Grewal etal.,44 using 1.25 mg bevacizumab (0.05 mL of 25 mg/mL)immediately after trabeculectomy, found improved bleb sur-vival at 6 months. This study, however, was conducted in asmall sample size, and follow-up was conducted over a short-period, nonrandomized, noncontrolled small case series of 12patients only.

Our findings show that bevacizumab, through the inhibitionof VEGF-A, reduces wound healing and scar formation at thelevel of the HTF through the combined inhibition of fibroblastproliferation and induced fibroblast cell death in addition to itseffect on angiogenesis. This, in conjunction with previousanimal studies39,43 and small case reports in humans, supportsthe notion that pharmacologic neutralization of VEGF-A withthe administration of bevacizumab offers a potentially safe andeffective adjunctive therapy to prevent the failure of trabecu-lectomy. It is enticing to consider that late bleb failure may betreated with anti-VEGF therapy in future studies to addressoptimization and dosage scheduling, safety profile, and long-term sequelae needed.

The findings from this study provide strong evidence thatVEGF-A is a key mediator for the development of conjunctivalvascularization and subconjunctival fibrosis. Furthermore, inparallel studies, we have also found that the combined deliveryof bevacizumab and 5-FU offers a synergistic elevated antifi-brotic effect when compared with their independent usageboth in vitro and in vivo (JGC, unpublished data, 2008). Thus,

bevacizumab potentially works synergistically with 5-FU todeliver a more profound effect on fibrosis. It is proposed thatthe antiangiogenic and antifibrotic effects of bevacizumabcould provide a magnified inhibitory effect on the woundhealing response.

In conclusion, to date anti–VEGF-A treatment has targetedpathologic angiogenesis for both systemic and ocular neovas-cular disorders. We have shown that bevacizumab, through theinhibition of VEGF-A, exerts a potent antifibrotic effect throughthe inhibition of fibroblast proliferation, induction of fibroblastcell death, and inhibition of cell-mediated collagen gel contrac-tion. These findings support the notion that adjunctive treat-ment with the VEGF-A inhibitor bevacizumab has the potentialto improve surgical outcomes after glaucoma filtration surgerywith greater safety and efficacy.

References

1. Addicks EM, Quigley HA, Green WR, Robin AL. Histologic charac-teristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol.1983;101:795–798.

2. Hitchings RA, Grierson I. Clinicopathological correlation in eyeswith failed fistulizing surgery. Trans Ophthalmol Soc U K. 1983;103(pt 1):84–88.

3. Skuta GL, Parrish RK 2nd. Wound healing in glaucoma filteringsurgery. Surv Ophthalmol. 1987;32:149–170.

4. Fluorouracil Filtering Surgery Study Group. Fluorouracil FilteringSurgery Study one-year follow-up. Am J Ophthalmol. 1989;108:625–635.

5. Kitazawa Y, Kawase K, Matsushita H, Minobe M. Trabeculectomywith mitomycin: a comparative study with fluorouracil. Arch Oph-thalmol. 1991;109:1693–1698.

6. Skuta GL, Beeson CC, Higginbotham EJ, et al. Intraoperative mit-omycin versus postoperative 5-fluorouracil in high-risk glaucomafiltering surgery. Ophthalmology. 1992;99:438–444.

7. Crowston JG, Akbar AN, Constable PH, Occleston NL, Daniels JT,Khaw PT. Antimetabolite-induced apoptosis in Tenon’s capsulefibroblasts. Invest Ophthalmol Vis Sci. 1998;39:449–454.

8. Lama PJ, Fechtner RD. Antifibrotics and wound healing in glau-coma surgery. Surv Ophthalmol. 2003;48:314–346.

9. Senger DR, Van de Water L, Brown LF, et al. Vascular permeabilityfactor (VPF, VEGF) in tumor biology. Cancer Metast Rev. 1993;12:303–324.

10. Fava RA, Olsen NJ, Spencer-Green G, et al. Vascular permeabilityfactor/endothelial growth factor (VPF/VEGF): accumulation andexpression in human synovial fluids and rheumatoid synovial tis-sue. J Exp Med. 1994;180:341–346.

11. Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growthfactor in ocular fluid of patients with diabetic retinopathy andother retinal disorders. N Engl J Med. 1994;331:1480–1487.

12. Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascu-lar membranes in age-related macular degeneration express vascu-lar endothelial growth factor. Invest Ophthalmol Vis Sci. 1996;37:1929–1934.

13. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–660.

14. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors.Nat Rev Cancer. 2002;2:727–739.

15. Shibuya M. Vascular endothelial growth factor-dependent and -in-dependent regulation of angiogenesis. BMB Rep. 2008;41:278–286.

16. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelialgrowth factor-induced angiogenesis suppresses tumour growth invivo. Nature. 1993;362:841–844.

17. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plusirinotecan, fluorouracil, and leucovorin for metastatic colorectalcancer. N Engl J Med. 2004;350:2335–2342.

18. Valachis A, Polyzos NP, Patsopoulos NA, Georgoulias V, MavroudisD, Mauri D. Bevacizumab in metastatic breast cancer: a meta-analysis of randomized controlled trials. Breast Cancer Res Treat.2010;122:1–7.


19. Andreoli CM, Miller JW. Anti-vascular endothelial growth factortherapy for ocular neovascular disease. Curr Opin Ophthalmol.2007;18:502–508.

20. Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and devel-opment of bevacizumab, an anti-VEGF antibody for treating can-cer. Nat Rev Drug Discov. 2004;3:391–400.

21. August DA, Serrano D, Poplin E. “Spontaneous,” delayed colon andrectal anastomotic complications associated with bevacizumabtherapy. J Surg Oncol. 2008;97:180–185.

22. Gordon CR, Rojavin Y, Patel M, et al. A review on bevacizumab andsurgical wound healing: an important warning to all surgeons. AnnPlast Surg. 2009;62:707–709.

23. Fung AE, Rosenfeld PJ, Reichel E. The International IntravitrealBevacizumab Safety Survey: using the Internet to assess drug safetyworldwide. Br J Ophthalmol. 2006;90:1344–1349.

24. Avery RL, Pearlman J, Pieramici DJ, et al. Intravitreal bevacizumab(Avastin) in the treatment of proliferative diabetic retinopathy.Ophthalmology. 2006;113:1695, e1–e15.

25. Avery RL, Pieramici DJ, Rabena MD, Castellarin AA, Nasir MA,Giust MJ. Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology. 2006;113:363–372, e5.

26. Bashshur ZF, Bazarbachi A, Schakal A, Haddad ZA, El Haibi CP,Noureddin BN. Intravitreal bevacizumab for the management ofchoroidal neovascularization in age-related macular degeneration.Am J Ophthalmol. 2006;142:1–9.

27. Bashshur ZF, Haddad ZA, Schakal A, Jaafar RF, Saab M, NoureddinBN. Intravitreal bevacizumab for treatment of neovascular age-related macular degeneration: a one-year prospective study. Am JOphthalmol. 2008;145:249–256.

28. Bashshur ZF, Haddad ZA, Schakal AR, Jaafar RF, Saad A, NoureddinBN. Intravitreal bevacizumab for treatment of neovascular age-related macular degeneration: the second year of a prospectivestudy. Am J Ophthalmol. 2009;148:59–65, e1.

29. Hasanreisoglu M, Weinberger D, Mimouni K, et al. Intravitrealbevacizumab as an adjunct treatment for neovascular glaucoma.Eur J Ophthalmol. 2009;19:607–612.

30. Mansour AM, Mackensen F, Arevalo JF, et al. Intravitreal bevaci-zumab in inflammatory ocular neovascularization. Am J Ophthal-mol. 2008;146:410–416.

31. Jonas JB, Spandau UH, Schlichtenbrede F. Intravitreal bevacizumabfor filtering surgery. Ophthalmic Res. 2007;39:121–122.

32. Kahook MY, Schuman JS, Noecker RJ. Needle bleb revision ofencapsulated filtering bleb with bevacizumab. Ophthalmic SurgLasers Imaging. 2006;37:148–150.

33. Khaw PT, Ward S, Porter A, Grierson I, Hitchings RA, Rice NS. Thelong-term effects of 5-fluorouracil and sodium butyrate on humanTenon’s fibroblasts. Invest Ophthalmol Vis Sci. 1992;33:2043–2052.

34. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPietroLA. Vascular endothelial growth factor mediates angiogenic activ-ity during the proliferative phase of wound healing. Am J Pathol.1998;152:1445–1452.

35. Cao Y, Linden P, Farnebo J, et al. Vascular endothelial growthfactor C induces angiogenesis in vivo. Proc Natl Acad Sci U S A.1998;95:14389–14394.

36. Cursiefen C, Chen L, Borges LP, et al. VEGF-A stimulates lym-phangiogenesis and hemangiogenesis in inflammatory neovascu-larization via macrophage recruitment. J Clin Invest. 2004;113:1040–1050.

37. Shibuya M, Claesson-Welsh L. Signal transduction by VEGF recep-tors in regulation of angiogenesis and lymphangiogenesis. Exp CellRes. 2006;312:549–560.

38. Hu DN, Ritch R, Liebmann J, Liu Y, Cheng B, Hu MS. Vascularendothelial growth factor is increased in aqueous humor of glau-comatous eyes. J Glaucoma. 2002;11:406–410.

39. Li Z, Van Bergen T, Van de Veire S, et al. Inhibition of vascularendothelial growth factor reduces scar formation after glaucomafiltration surgery. Invest Ophthalmol Vis Sci. 2009;50:5217–5225.

40. Lee KS, Park SJ, Kim SR, et al. Inhibition of VEGF blocks TGF-beta1production through a PI3K/Akt signalling pathway. Eur Respir J.2008;31:523–531.

41. Li ZD, Bork JP, Krueger B, et al. VEGF induces proliferation,migration, and TGF-beta1 expression in mouse glomerular endo-thelial cells via mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase. Biochem Biophys Res Commun. 2005;334:1049–1060.

42. Wilgus TA, Ferreira AM, Oberyszyn TM, Bergdall VK, Dipietro LA.Regulation of scar formation by vascular endothelial growth factor.Lab Invest. 2008;88:579–590.

43. Memarzadeh F, Varma R, Lin LT, et al. Postoperative use of bev-acizumab as an antifibrotic agent in glaucoma filtration surgery inthe rabbit. Invest Ophthalmol Vis Sci. 2009;50:3233–3237.

44. Grewal DS, Jain R, Kumar H, Grewal SP. Evaluation of subconjunc-tival bevacizumab as an adjunct to trabeculectomy a pilot study.Ophthalmology. 2008;115:2141–2145, e2.

45. Kahook MY, Schuman JS, Noecker RJ. Intravitreal bevacizumab ina patient with neovascular glaucoma. Ophthalmic Surg LasersImaging. 2006;37:144–146.

46. Kitnarong N, Chindasub P, Metheetrairut A. Surgical outcome ofintravitreal bevacizumab and filtration surgery in neovascular glau-coma. Adv Ther. 2008;25:438–443.


antifibrotic activity of bevacizumab on human tenon's fibroblasts in vitro

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