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INTRODUCTION

Age-related macular degeneration (AMD) is the main cause for legal blindness in the elderly in the industrial-ized world. Pathological angiogenesis is the underlying cause of the exsudative form of AMD. Since vascular

endothelial growth factor (VEGF) has a cardinal affect on the angiogenesis process,1 The development of VEGF antagonists introduced a new treatment regimen, which led to much better long-term results.2 In 2006, Ranibizumab (Lucentis), an antibody fragment devel-oped against all fragments of VEGF, was approved by the FDA for treating AMD, following the MARINA2 and the ANCHOR3 studies which showed both a stabi-lizing effect in 90% of the patients and a beneficial effect in 30% of the patients.

Current Eye Research, 35(9), 835–841, 2010Copyright © 2010 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713683.2010.489727

ORIGINAL ARTICLE

Influence of Non-Toxic Doses of Bevacizumab and Ranibizumab on Endothelial Functions and

Inhibition of Angiogenesis

Aya Barzelay1, Anat Lowenstein2, Jacob George1, and Adiel Barak2

1Vascular Biology Lab, Tel Aviv Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel2Department of Ophthalmology, Tel Aviv Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv,

Israel

ABSTRACT

Purpose: Ranibizumab (Lucentis) is an antibody fragment developed against all fragments of vascular endothelial growth factor (VEGF) that was approved by the FDA for treating age-related macular degeneration (AMD). Bevacizumab, a full-length anti-VEGF antibody approved for use in colon cancer, is non-FDA approved at this time but it is widely used for treating AMD. The purpose of this study was to compare the influence of Bevacizumab and Ranibizumab on angiogenesis in an in vitro model.Methods: A model consisting of H5V cells derived from murine hearts capillary endothelial cells (ECs) was used. The H5V cells were treated with three concentrations of Bevacizumab and Ranibi-zumab (0.125 mg/mL, 0.25 mg/mL, and 0.50 mg/mL) for 24 hr before all experiments. The effects of Bevacizumab and Ranibizumab on EC proliferation were compared by 3H-thymidine incorporation essay. Toxic effects and the safety of each drug in clinical concentrations were assessed by annexin 5 staining. The effects of the drugs on ECs functions were assessed by their ability to adhere to fibronectin and by evaluation of the cells’ tube formation capacity on matrigel.Results: Both Bevacizumab and Ranibizumab equally suppressed the adhesive properties of ECs to fibronectin, and similarly inhibited ECs’ proliferation capacity in a dose-dependent manner. Both Bevacizumab and Ranibizumab inhibited the ECs’ tube formation capacity on matrigel, and were equally safe.Conclusions: Ranibizumab and Bevacizumab at low, non-toxic doses similarly inhibit several prop-erties of the angiogenesis process. Inhibition of ECs adhesion to fibronectin and tube formation capacity does not seem to be directly related to the anti-angiogenic effects as indicated by inhibi-tion of VEGF. Further studies for delineating the exact mechanism of action of Ranibizumab and Bevacizumab in angiogenesis are warranted.

KEYWORDS: Age related macular degeneration; Angiogenesis; Anti-angiogenic therapy; Choroidal neovascularization; Retina

Received 03 July 2009; accepted 25 April 2010

Correspondence: Adiel Barak, M.D., Department of Ophthalmol-ogy, Tel Aviv Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. E-mail: omadb@post.tau.ac.il

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Ranibizumab is highly effective but considerably expensive. In contrast, Bevacizumab, a full-length anti-VEGF antibody approved for use in colon cancer,4 costs about 1/4 of the price of ranibizumab. Bevaci-zumab is non-FDA approved at this time, but still, it is widely used for treatment of AMD.5 Bevacizumab was described in case reports and retrospective studies as having good effectiveness and tolerability, but it has never been tested in a clinical phase III trial.6

The objective of the current study was to compare VEGF inhibitors in their ability to prevent various aspects of angiogenesis in a quantifiable, reproduc-ible in vitro setting. To date, only limited experimental data and studies comparing the effects of Bevacizumab and Ranibizumab on porcine retina, retinal pigment epithelium, choroid organ culture and retinal pig-ment epithelium cell culture, have been reported.7,8 The model we chose was designed to compare the effects of VEGF inhibitors on ECs’ proliferation by 3H-thymidine incorporation essay, and to evaluate the safety of these VEGF inhibitors by annexin 5 staining. We further examined functional angiogenesis assays by investigating the effect of VEGF inhibitors on the ability of ECs to adhere to fibronectin as well as on their tube formation capacity on matrigel.

METHODS

Cell Culture

H5V cells derived from murine hearts capillary endothelial cell line9 were grown in 37°C, and 8% CO2 in Dulbecco’s modified Eagle medium (DMEM)/F12 (Biological Industries, Beit Haemek, Israel), supple-mented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% FCS.

In the current study we have chose to use H5V cell line and not the human umbilical vein endothelial cells (HUVEC). In our laboratory we have tremendous experience regarding both cell lines.10–12 Our previous experience with both cell lines shows that HUVEC cell line is too sensitive, and cells do not survive more then three splits. Hence, repeating an experiment in the exact same conditions is impaired. Moreover, H5V cell line is more potent and cells survive several splits. When we perform experiment with H5V cell line we know that all repeats were conducted under same conditions when taking in consideration the viability of the cells.

Exposure to Bevacizumab and Ranibizumab

Before all experiments, H5V cells were treated for 24 hr with three standard clinical dosage range

concentrations of Bevacizumab or Ranibizumab: 0.125 mg/mL, 0.25 mg/mL, and 0.50 mg/mL.

These concentrations were prepared by using serial dilutions of the drug in the respective serum-free cul-ture medium. The solution of the drug mixed with media was then directly added to the cells in order to obtain a uniform concentration of drug throughout the well of the tissue culture plate. In addition to Bevaci-zumab and Ranibizumab, the H5V cells were treated with 10 ng/ml of hVEGF (cytolab).

Control Groups

All experiments were compared to both positive and negative controls which were consisted as follows: Positive controls were H5V cells which were treated with hVEGF alone and no drugs. Negative controls were H5V cells which were not treated with neither VEGF or with Bevacizumab and Ranibizumab.

Results are normalized to positive controls and presented as folds of positive controls.

Proliferation Assay

The proliferation index was determined by first syn-chronizing the cells at the G1-S boundary by culturing cells for 24 hr in a serum free medium. Then, 4 * 10 ^ 3 H5V cells/well were seeded in 96-well plates. H3-thymidine (1 μCi/well) was added for 16 hr before proliferation was assayed by scintillation counting (β counter).

Adhesion Assay

In order to evaluate adhesion to fibronectin, 10 ^ 4 H5V cells/per well were seeded in 96-well plates coated with fibronectin for 30 min. Non-adherent cells were then washed away and the adherent cells were stained with XTT-based colorimetry (Biological Industries, Beit Haemek, Israel). Optical density at 450 nm, pro-portional to viable cell numbers, was measured using an ELISA reader. Fibronectin-coated wells served as background.

Assay of Tube Formation Capacity on Matrigel

Twenty-four hours before performing the assays, Matrigel (BD Matrigel™ Matrix Growth Factor Reduced, BD Biosciences cat. No. 354230) was stored at 4°C and transferred to ice immediately before coating the 24-well plates. Then, H5V (1.5 × 10 ^ 4

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cells/per well) were seeded in these plates. The cells’ tube formation capacity on matrigel was evaluated after 16 hr by phase-contrast microscopy (×40) and scored as described elsewhere8: 0, individual cells, well separated; 1, cells begin to migrate and align themselves; 2, capillary tubes visible, no sprouting; 3, sprouting of new capillary tubes visible; 4, closed polygons begin to form; 5, complex mesh-like struc-tures develop.

Annexin Staining

The cells were washed twice with cold PBS and then re- suspended in 85 µl 1X Binding Buffer (MEBCYTO apoptosis kit® Cat. No. 4700) at a concentration of ∼2 × 105 cells/ml. Ten µl of annexin V-FITC and 5 µl of propidium iodide were added to 85 µl cell suspension and incubated for 15 min at room tem-perature. The stained cells were analyzed by flow cytometry using a single laser-emitting excitation light at 488 nm.

Statistical Analysis

Every experiment was independently repeated at least three times. Significance was calculated with t-tests and graphing software (Sigma Plot; Systat, Chicago, Illinois, USA). P < 0.05 was considered significant. Results are expressed as mean ± SD.

All the results represent data of one representative experiment; the experiments were repeated twice with similar statistical significance.

RESULTS

Bevacizumab and Ranibizumab Similarly Inhibited EC Proliferation Capacity in a Dose-Dependent Manner

Given that proliferation is a functional property that characterizes activated angiogenic ECs, we examined the effect of treatment by Bevacizumab and by Ranibi-zumab on the cells’ proliferation capacity. Both Bevaci-zumab and Ranibizumab inhibited ECs’ proliferation, with no significant difference in the extent of this inhibitory competence between both drugs, as com-pared to positive control of ECs that were treated with VEGF alone. All treated cell groups showed a dose-de-pendent decrease in proliferation compared to controls (B1 mg/mL 0.9 ± 0.35; R1 mg/mL 0.89 ± 0.24; B2 mg/mL 0.82 ± 0.25; R2 mg/mL 0.69 ± 0.33; B3 mg/ mL 0.45 ± 0.12; R3 mg/mL 0.49 ± 0.13) (Figure 1).

Bevacizumab and Ranibizumab Equally Suppressed the ECs’ Adhesive Properties

Fibronectin is a component of the extra cellular matrix and the ligand of integrins expressed in ECs.13,14 VEGF is a known factor influencing Ecs’ adhesion properties.15 Moreover, an increased EC adhesion capacity plays a crucial role in EC growth and acti-vation, particularly during new vessel sprouting.16 Herein, we assessed the ability of the H5V EC line to adhere to fibronectin following treatment consisting of either Bevacizumab or Ranibizumab in combina-tion with VEGF. We therefore used a previously described attachment assay17 to study the influence of Bevacizumab and Ranibizumab on EC adhe-sion to fibronectin. Either Bevacizumab or Ranibi-zumab were added for 24 hr in three concentrations: 0.125 mg/mL, 0.25 mg/mL, and 0.50 mg/ mL. The control group was comprised of ECs which received only the pro-angiogenic cytokine VEGF. The ECs which had been treated with either Bevacizumab or Ranibizumab showed decreased adhesive capac-ity, with no significant difference between treated groups; as compared to positive control (results are normalized to positive controls and presented as folds of controls: B1 0.75 ± 0.05; R1 0.76 ± 0.05, B2 0.76 ± 0.04; R2 0.75 ± 0.02, B3 0.81 ± 0.17; R3 0.76 ± 0.02; p < 0.05) (Figure 2).

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FIGURE 1 The effect of Bevacizumab and Ranibizumab on endothelial cells (ECs) proliferation. Both Bevacizumab and Ranibizumab inhibited ECs’ proliferation with no significant difference in the extent of this inhibitory ability as compared to control. ECs were first synchronized at the G1-S boundary by culturing cells for 24 hr in a serum-free medium, then, 4 * 10 ^ 3 H5V cells/well were seeded in 96-well plates. VEGF, Bevacizumab, Ranibizumab, and 3H-thymidine (1 μCi/well) were added for 16 hr before proliferation was assayed by scin-tillation counting (β counter). At each concentration, student t-test was performed between Bevacizumab- and Ranibizum-ab-treated cells. Difference between drugs at each concentra-tion was not significant. B—Bevacizumab; R—Ranibizumab at 3 concentrations: 1: 0.125 mg/mL, 2: 0.25 mg/mL, and 3: 0.50 mg/mL. – Negative control, + Positive control. Y-Axis: Folds of positive control.

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Both Bevacizumab and Ranibizumab Inhibited ECs’ Tube Formation Capacity on Matrigel

The Matrigel assay is a morphogenesis assay that measures cell functions necessary for angiogenesis.18 In order to align to capillary-like structures in the Matrigel’s basement membrane-like matrix, ECs must adhere to and move on the extra cellular matrix, a process which is mainly dependent on various integ-rins.19 We next explored the effect of Bevacizumab and Ranibizumab on the ability of ECs to form capillary-like tubules on Matrigel-coated plates. The ECs that had been treated with either Bevacizumab or Ranibizumab showed poor tube formation capacity compared to positives controls which were treated only with VEGF. Bevacizumab treatment resulted in better attenuation of ECs to form tubes, at all three concentrations in comparison to Ranibizumab treatment. While all con-centration of Bevacizumab treatment showed similar inhibition effect, there was evidence of dose-dependant reduction in Ranibizumab effect on ECs tube formation (B1 0.26 ± 0.1; B2 0.33 ± 0.1; B3 0.32 ± 0.09; R1 0.55 ± 0.25; R2 0.48 ± 0.15; R3 0.39 ± 0.15; p < 0.05) (Figure 3).

No Variation in Safety of Treating ECs with Bevacizumab or Ranibizumab

We performed flow cytometric analysis in order to determine whether either Bevacizumab or Ranibi-zumab had an advantage in regard with the amount of cell toxicity (i.e., any amount of cell toxicity, annexin

V detection of dead cells, necrosing cells, and cells in early stages of apoptosis). Both drugs were safe in clinical dosage range, as evidenced by our results that no drug showed increase of EC apoptotic cells with compare to controls (Figure 4). (All treated groups showed an average of 0.99 of control.)FIGURE 5

DISCUSSION

AMD is a degenerative disease in which in an estimated 70% of cases,20 is characterized by the abnormal growth of local choroidal blood vessels. In order to form new blood vessel, endothelial cells are activated to prolifer-ate, to migrate into the forming vessel, and to adhere to the extra cellular matrix and to adjacent cells, while the whole process is conducted through paracrine activity by the secretion of angiogenic cytokines.21 These functional properties were shown to be dependent in VEGF.15,22,23 Hence, in order to compare the inhibitory affect both Bevacizumab and Ranibizumab has on Ecs’ angiogen-esis; we examined the above functional properties.

The findings of the current in vitro study showed that both Bevacizumab and Ranibizumab equally sup-pressed the adhesive properties of ECs to fibronectin, similarly inhibited the cells’ proliferation capacity in a dose-dependent manner, inhibited the cells’ tube formation capacity on Matrigel, and were equally safe for treating ECs.

First, we sought to determine whether Bevacizumab and Ranibizumab at clinically used concentrations have toxic effects on proliferating ECs. Again, as expected, both drugs similarly inhibited the ECs’ proliferation capacity in a dose-dependent manner, and had no sig-nificant toxic effects on proliferating cells even at the lowest dose examined. We did not try to establish the LD50 concentration for either of the two compounds in the current work. In another set of examinations, we found (as previously described7) that neither compound given in clinically used concentrations had toxic effects on non-proliferative ECs, thus demonstrating their safety for use in contact with non-proliferative tissues.

As for adhesive capacity of ECs, the ECs that were treated with either Bevacizumab or Ranibizumab showed decreased adhesive capacity, similar to nega-tive control ECs, which underwent no treatment at all. There was no demonstrable influence on adhesion to fibronectin with increasing dosage (from 0.125 mg/mL to 0.50 mg/mL). This indicates that both Bevacizumab and Ranibizumab might influence complex transcrip-tion or translation processes leading to altered integrin-mediated adhesion, and that even low doses of both compounds are capable of causing these effects. The exact mechanisms through which VEGF antagonists influence VEGF must be elucidated. VEGF expres-

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FIGURE 2 The effect of Bevacizumab and Ranibizumab on endothelial cells (ECs) adhesion capacity. 10 ^ 4 H5V cells/per well were seeded in 96-well plates coated with fibronectin for 30 min. Non-adherent cells were then washed away and the adherent cells were stained with XTT-based colorimetry. Both drugs were given to ECs for 24 hr prior to the experiment, in three concentrations, 0.125 mg/mL, 0.25 mg/mL, and 0.50 mg/mL. The control groups consisted of ECs that received VEGF alone (positive control), and ECs that didn’t receive VEGF or any drug (negative control). Treatment with both drugs decreased ECs adhesive capacity, with no significant difference between treated groups. B—Bevacizumab; R—Ranibizumab at 3 concentrations: 1: 0.125 mg/mL, 2: 0.25 mg/mL, and 3: 0.50 mg/mL; – Negative control, + Positive control. Y-Axis: Folds of positive control.

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sion might be modulated directly through unknown pathways, such as internalization and consequent intracellular effects. Bevacizumab has indeed been shown to be internalized by RPE cells.24 In addition, the inhibition of VEGF binding to its receptor might cause feedback mechanisms that influence VEGF expression. Different VEGF isoforms have been shown to regulate the expression of other isoforms.7,25,26

We next demonstrate that the ECs’ alignment needed to form branching anastomosing tubes when seeded

on matrigel, was significantly reduced (Figure 3). The remaining cells were mostly isolated and spherical. The results obtained at low doses were comparable to those obtained for 1 pM vinblastine, as described by Vacca et al.17 and for 5 µM temozolomide, as described by Kurzen et al.27 VEGF was shown to be crucial for endothelial cells tube formation capacity.15,22 Again, these findings suggest a VEGF inhibiion activity of both compounds that may prevent the angiogenic process in ECs in a yet undefined pathway.

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FIGURE 3 The effect of Bevacizumab and Ranibizumab on endothelial cells’ (ECs) tube forma-tion capacity. ECs were exposed to VEGF, Bevacizumab, and Ranibizumab 24 hr prior and dur-ing the experiment. Twenty-four well plates were coated with matrigel, and then H5V (1.5 × 10 ^ 4 cells/per well) were seeded in these plates. The cells’ tube formation capacity on matrigel was evaluated after 16 hr by phase-contrast microscopy (×40) (A), and scored 1–5 (0, individual cells, well separated; 1, cells begin to migrate and align themselves; 2, capillary tubes visible, no sprouting; 3, sprouting of new capillary tubes visible; 4, closed polygons begin to form; 5, com-plex mesh-like structures develop). Each group was scored 1–5 and was compared to a positive control group that consisted H5V cells that were treated with hVEGF alone and no drugs (data is represented as folds of control) (B). Bevacizumab treatment resulted in better attenuation of ECs to form tubes, at all three concentrations (B1–B3) in comparison to Ranibizumab treatment. B—Bevacizumab; R—Ranibizumab at 3 concentrations: 1: 0.125 mg/mL, 2: 0.25 mg/mL, and 3: 0.50 mg/mL. – Negative control, + Positive control. Y-Axis: Tube formation index/high power field. Folds of positive control.

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Inhibition of VEGF likely results in both inhibition of proliferation and induction of cell apoptosis. In the current study, we sought to compare the toxic effect Bevacizumab and Ranibizumab has on endothelial cells. In order to do so, we have examined the apop-tosis rates of treated cells without adding another pro-apoptotic factor (H2O2 for example) to the system beside the examined drugs. Herein we assume that the inhibition of VEGF that was caused by the drugs was indeed sufficient to inhibit proliferation, as indicated in Figure 1, but was not dramatic enough in to cause vast cell apoptosis, as indicated in Figure 4, a result that may suggest on the drugs’ safety.

Our manuscript has several limitations: H5V cells were used, which have both murine and bovine ori-gin. However both Avastin and Lucentis are highly humanized, e.g., Avastin has only 7% murine origin and 93% human origin.3 The question remains whether the result we found are related to the non toxic effect of Avastin or Luecentis or to the limitation of the assays which were used. Therefore, the conclusion could be that both drugs do not have any effect in the assays used. We believe that the drugs do have effect, because the positive controls show a significant change in reac-tion to the treatment. Furthermore, the VEGF which was used in the study was of human origin, and cell did react to the addition of human VEGF. Yet our experimental model is not ideal, and the results must be taken with caution and if possible repeated with human endothelial cells.

Our findings that Bevacizumab and Ranibizumab have similar anti-angiogenic effects are not surpris-ing, considering that both compounds are antibodies

and antibody fragments of the same murine precur-sor.28 Several studies have focused on the comparison between Bevacizumab and Ranibizumab regarding the drug’s anti angiogenic properties.7,29 Our results are consistent with those of previous in vitro works which showed that Bevacizumab and Ranibizumab, given in clinical doses, are equally potent in neutralizing VEGF.7 Ranibizumab is a 48 kDa Fab fragment that can easily penetrate the retina, unlike a full length antibody.30 Our model could not detect any difference in penetration factors between Ranibizumab and Bevacizumab, and this may be crucial when considering their use in a clinical setting.

Several questions remain unanswered in our study and needs to be studied in future in vivo trials. First, the questions whether there are any differences in inhibitory competence of both studied drugs in vivo, where penetration differences may exist and therefore dose effects on target choroidal endothelial cells may come into play. Second, the Fc portion on the Beva-cizumab molecule may contribute to immunological effects which may alter the local clinical effects and additionally alter the metabolism of the drug that would then lead to dose differences between the two drugs at any time point.

Currently several clinical trails are comparing the effects of Bevacizumav versus Ranibizumab in patients with age related macular degeneration. CATT (Com-parison of Age-Related Macular Degeneration Treat-ments Trials: Lucentis-Avastin Trial)31 in the US is the bigger one, but searching the US clinical trail registry reveals at least 10 different trails which examine the effects of Bevacizumav vs. Ranibizumab.32 Within few years, the clinical comparison of the two drugs will be elucidated, but until then the in vitro data adds valu-able information for better understanding and treat-ment of the AMD patients.

In summary, the present study demonstrated that Ranibizumab and Bevacizumab both inhibited several aspects of the angiogenesis process at low, non-toxic doses. Our data suggests that no significant advantage can be related to either drug in vitro, since inhibitory competences of both drugs seemed rather equivalent. Inhibition of EC adhesion to fibronectin and tube for-mation capacity on matrigel did not seem to be directly related to the observed anti-angiogenic effects via VEGF inhibition. Further studies are needed to delin-eate the precise mechanism of action of Ranibizumab and Bevacizumab in angiogenesis.

ACKNOWLEDGMENT

The authors with to thank Esther Eshkol for editorial assistance.

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FIGURE 4 Annexin V-FITC and propidium iodide staining of endothelial cells (ECs). Twenty-four hours after treatment with VEGF, Bevacizumab, and Ranibizumab, annexin V-FITC and propidium iodide were added to cell suspensions and incubated for 15 min at room temperature. The stained cells were analyzed by flow cytometry using a single laser-emitting excitation light at 488 nm. Non-apoptotic cells at each group are presented as folds of non-apoptotic cells in the positive control group. Both drugs were equally safe in a clinical dos-age range. B—Bevacizumab; R—Ranibizumab at 3 concen-trations: 1: 0.125 mg/mL, 2: 0.25 mg/mL, and 3: 0.50 mg/mL. – Negative control, + Positive control. Y-Axis: Folds of positive control.

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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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