Comparative Evaluation of Apoptotic Activity in Photoreceptor Cells After Intravitreal Injection of Bevacizumab and Pegaptanib Sodium in Rabbits

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Comparative Evaluation of Apoptotic Activity in Photoreceptor Cells after Intravitreal Injection of Bevacizumab and Pegaptanib Sodium in Rabbits

Berrin Avci,1 Remzi Avci,2 U¨mit U¨beyt Inan,3 and Berkant Kaderli2

PURPOSE. To evaluate quantitatively the apoptotic activity after Angiogenesis has an important role in choroidoretinal vascular intravitreal injections of pegaptanib sodium and bevacizumab diseases such as proliferative diabetic retinopathy, age-related in the rabbit retina. macular disease (ARMD), and retinopathy of prematurity. The METHODS. Different doses of bevacizumab (0.25, 0.625, 1.25, effects of VEGF antagonists on ocular neovascularization have raised new hope for the treatment of proliferative vascular and 2.5 mg) and pegaptanib sodium (0.15, 0.3, and 0.6 mg) 3,4 were injected intravitreally in 48 rabbits. The eyes were enu- diseases. cleated at different times for early studies at day 14 and for late Bevacizumab is an anti-VEGF humanized monoclonal anti- studies at 3 months after a single injection or at 3 months, with body that binds to all biological active isoforms of VEGF. It is 1 injection in each of the 3 months (day 90). The time course the first antiangiogenic agent to be approved by the FDA for and dose–response of photoreceptor cells in the rabbit retina the treatment of metastatic colorectal cancer in conjunction after intravitreal injection of bevacizumab or pegaptanib so- with chemotherapy. Although bevacizumab cannot theoreti- dium were examined by histologic analysis with hematoxylin cally penetrate all retinal layers efficiently because of its large and eosin (H&E) staining, caspase-3 and -9 immunostaining, size, it penetrates to deeper retinal layers, and clinically prom- ising results have been reported after intravitreal application in and in situ terminal-deoxynucleotidyl transferase-mediated bi- 5–8 otin-deoxyuridine triphosphate nick-end labeling (TUNEL) of the treatment of subretinal neovascularization. Despite on- DNA fragments of paraffin-embedded sections. going clinical trials with low-dose intravitreal bevacizumab application in various neovascular ocular disorders, its ocular RESULTS. No sign of retinal toxicity was seen in H&E stained safety is still an issue for research. Several preclinical experi- histologic sections of eyes that had received bevacizumab or mental toxicity studies have reported the histopathologic ef- pegaptanib sodium. Nuclear DNA fragmentation in the outer fects of bevacizumab on retinal cells, retinal neovascular mem- retinal layers shown by the TUNEL method was evident in the branes, and capillaries in paraffin-embedded sections5–9 or high-dose groups (55.3% with 1.25 mg and 64.5% with 2.5 mg organotypic culture,10–12 as well as in ultrastructural evalua- bevacizumab, and 48.5% with 0.6 mg pegaptanib sodium) at 14 tions.5,13,14 days and also in the clinical dose groups (49.8% with three Pegaptanib sodium, an anti-VEGF RNA aptamer specific to injections [1 each month] of 0.625 mg bevacizumab and 44.3% the VEGF165 isoform, has been used for the treatment of all with 0.15 mg pegaptanib sodium) at 90 days. The ratios of types of neovascular ARMD.15–17 Clinical studies have shown TUNEL-positive cells in physiologic saline and the sham-control that the inhibition of VEGF by intravitreal pegaptanib sodium groups were 32.3% and 21%, respectively. results in the stabilization or improvement of vision in patients CONCLUSIONS. Intravitreal injection of bevacizumab and pe- with neovascular ARMD; and, unlike bevacizumab, it has been gaptanib sodium caused a significant increase in apoptotic produced for intraocular application.15,18–20 However, there activity in rabbit photoreceptor cells. However, although bev- are limited preclinical studies associated with the intravitreal acizumab caused increasing apoptotic activity at higher doses, administration of pegaptanib sodium in vivo21 and in vitro.10,12 similar dose-dependent adverse effects were not evident for In our previous study, we investigated early and late-term pegaptanib sodium. (Invest Ophthalmol Vis Sci. 2009;50: retinal toxicity of bevacizumab by light and electron micro- 3438–3446) DOI:10.1167/iovs.08-2871 scopic study in rabbit eyes and found ultrastructural changes in the mitochondria in the inner segments of the photoreceptors, ascular endothelial growth factor (VEGF) has angiogenic suggesting increased apoptotic activity. The results of the im- Vand mitogenic activity with specificity to endothelial cells munohistochemical study with caspase-3, caspase-9, and bax/ in vivo. It also possesses the physiological function of increas- bcl-2 staining supported apoptotic activity in photoreceptor ing the permeability of capillary microvessels to different mac- cells.13 romolecules, resulting in blood–tissue barrier breakdown.1,2 In the present study, we investigated the quantitative evaluation of apoptotic activity, mainly by using the TUNEL (in situ terminal–deoxynucleotidyl transferase-mediated biotin-deoxy- From the Departments of 1Histology and Embryology and 2Oph- uridine triphosphate nick-end labeling of DNA fragments) thalmology, Uludag University School of Medicine, Bursa, Turkey; and method in rabbit retinal cells after application of intravitreal the 3Department of Ophthalmology, Kocatepe University School of bevacizumab and also pegaptanib sodium with different dose Medicine, Afyon, Turkey. schedules. Supported in part by Pfizer Ltd. Submitted for publication September 13, 2008; revised December 23, 2008, and January 31, 2009; accepted May 6, 2009. MATERIALS AND METHODS Disclosure: B. Avci, Pfizer (F); R. Avci, Pfizer (F); U¨.U¨ . Inan, Pfizer (F); B. Kaderli, Pfizer (F) Full-length humanized rhuMAB vascular endothelial growth factor The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- (VEGF) antibody (bevacizumab, Avastin; Genentech/Roche, San Fran- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. cisco, CA) and an RNA aptamer directed against VEGF-165 (pegaptanib Corresponding author: Berrin Avci, Department of Histology and sodium, Macugen; Pfizer Ltd., New York, NY), the VEGF isoform Embryology, Uludag University Medical Faculty, 16059 Bursa, Turkey; primarily responsible for pathologic ocular neovascularization and vas- [email protected]. cular permeability were used in the study.

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TABLE 1. Treatment Groups

Pegaptanib Sodium Bevacizumab (25 mg/mL) (3.47 mg/mL) Saline Control Dose Dose Groups (mg/␮L) Groups (mg/␮L) Group Dose Group Dose

1 (day 14) 0.25/10 7 (day 14) 0.15/45 12 50 ␮L 13 None 2 (day 14) 0.625/25 8 (day 14) 0.3/90 3 (day 14) 1.25/50 9 (day 14) 0.6/180 4 (day 14) 2.5/100 10 (month 3) 0.15/45 5 (month 3) 0.625/25 11 (month 3) 0.15/45 ϫ 3 6 (month 3) 0.625/25 ϫ 3

n ϭ 4 eyes for each of the drug, saline injection, and control groups. Group 13 was the noninjected sham control.

Animals The pupils were fully dilated with 2.5% phenylephrine hydrochloride and 1% tropicamide. After ocular surface anesthesia with a topical The clinical and experimental protocols were approved by the Animal instillation of proparacaine (Alcain; Alcon, Fort Worth, TX) to reduce Care and Use Committee of Uludag University and are in accordance the animal’s discomfort, the eyes were washed with several drops of with the guidelines set forth by the ARVO Statement for the Use of 5% povidone iodide. A 27-gauge standard disposable needle for bev- Animals in Ophthalmic and Vision Research and the institutional guide- acizumab injection or 27-gauge needle of prefilled syringe for pe- lines regarding animal experimentation in ophthalmic and vision re- gaptanib sodium was introduced into the vitreous cavity 1.0 to 1.5 mm search. The eyes used in the study were taken from adult male albino posterior to the superotemporal limbus, and the needle tip was di- rabbits obtained from the Experimental Animal Breeding and Research rected into the midvitreous under direct visualization with external Centre of Uludag University. The animals were fed standard laboratory illumination of an indirect ophthalmoscope. One volume of the drug food and allowed free access to water in an air-conditioned room with solution was then slowly administered according to the group alloca- a 12-hour light–dark cycle. tion of the animal. The needle was held in place for 10 seconds before Forty eight adult male rabbits weighing 1.8 to 2.5 kg each on the withdrawal, to prevent reflux from the entry site. The central retinal day before drug administration were assigned to 11 study groups (n ϭ artery was observed by indirect ophthalmoscopy to ensure patency 4 in each group) and one control group (n ϭ 2, 4 eyes) for sham after each injection. Ofloxacin eye drops (0.3% Exocin; Allergan, Ir- control and one saline-injection control group (n ϭ 2, 4 eyes; Table 1). vine, CA) were applied to the eyes of the experimental group animals, By assuming rabbit vitreous volume as 2.5 mL and human vitreous as immediately after the procedure. approximately 5 mL, the doses of 0.625 mg (25 ␮L, 25 mg/mL) bevacizumab and 0.15 mg (45 ␮L, 3.47 mg/mL) pegaptanib sodium were regarded as clinical doses because the doses of 1.25 and 0.3 mg, Toxicity Assessment respectively, are used clinically in humans.22 Early (14 days) groups Clinical Observations. Study eyes were clinically examined, were assigned doses below (0.25 mg) and above (1.25 and 2.5 mg) the first before the injection, then 3 days after, and finally at the end of the clinical doses for eyes receiving bevacizumab. study. At follow-up the pupils were dilated with tropicamide eye Groups 1 to 4 received 0.25, 0.625, 1.25 and 2.5 mg intravitreal drops. According to a prospective protocol, the following parameters single-dose bevacizumab, respectively, and groups 7 to 9 received were recorded: corneal clarity, transparency, appearance of the lens 0.15, 0.3, and 0.6 mg intravitreal single-dose pegaptanib sodium, re- and retina, conjunctival reaction, proteinaceous ray, and cells in the spectively. Groups 1 to 4 and 7 to 9 were labeled as early groups and anterior and posterior segments of the eye. scheduled to be killed on day 14. Right eyes were assigned as study At the baseline, immediately and 3 days after intravitreal injection, eyes. Group 5 received a single 0.625-mg dose of intravitreal bevaci- all study and control eyes were examined by indirect ophthalmoscopy zumab and group 6 received one injection of 0.625-mg intravitreal with a 20-D aspherical lens for clear and sharp imaging of the retina bevacizumab in three consecutive months. Group 10 received a and to exclude any possible disease in the vitreous, retina, and cho- 0.15-mg single dose pegaptanib sodium and group 11 received three roids. monthly injections of 0.15 mg intravitreal pegaptanib sodium. Groups The appearance of the conjunctiva and cornea in addition to the 5, 6, 10, and 11 were labeled as late groups and scheduled to be killed examination of the anterior chamber and anterior vitreous for evalua- at the end of month 3. Group 12 (both eyes of two rabbits) received tion of cellular reaction and flare were inspected by handheld slit lamp 0.05 mL physiologic saline (balanced salt solution, BSS Plus; Alcon, Fort under the highest magnification. Worth, TX) intravitreally for the saline control. Group 13 consisted of Histopathologic Analysis. After the final clinical examination both eyes of two rabbits that did not receive any injection, but were on day 14 (early groups) or by month 3 (late groups), the animals were exposed to the press of a needle tip for sham control. After the rabbits killed by intraperitoneal injection of a lethal overdose of pentobarbital were killed, the study and control eyes were enucleated and preserved (100 mg/kg body weight). The eyes were enucleated with careful for light microscopic evaluation. manipulation to preserve globe integrity. Each eye was immediately placed in neutral formalin solution for light microscopic examination. Procedure The eyes were fixed in neutral formalin solution for 2 days and Intravitreal injections were performed after a baseline ophthalmic immersed in 30% sucrose solution for easy dissection for another 48 examination with a handheld slit lamp biomicroscope (Kowa Ltd., hours. Then, the enucleated eyes were bisected vertically, dehydrated Nagoya, Japan), determination of intraocular pressure (Tono-pen XL; in a series of 95% ethanol, and embedded in paraffin. The sections Medtronic, North Jacksonville, FL), and funduscopy by indirect oph- were obtained from slices through the whole globe oriented along the thalmoscopy. Before all intravitreal injections, the rabbits were anes- optic nerve and medullary ray. Sagittal retinal sections of 5-␮m thick- thetized by intramuscular injection (0.5 mL/kg body weight) of a ness were collected on poly-L-lysine–coated microscopic slides for mixture containing ketamine hydrochloride (25 mg/kg) and xylazine H&E staining, immunohistochemical analysis with caspase-3 and -9 hydrochloride (2 mg/kg) solution. staining, and TUNEL assay. All slides were examined by light micro-

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scope (BX-50; Olympus, Tokyo, Japan) and photographed with a digital Finally, the sections were stained with DAB chromogen and cover- video camera (DP-71 CCD; Olympus). slipped. In the negative control experiment, sections were processed In Situ TUNEL Protocol. DNA nick end-labeling was per- using the same protocol as above, except that the primary antibody formed with an apoptosis detection kit (ApopTag Plus Peroxidase In was replaced by blocking serum. Positive controls were formaldehyde Situ Apoptosis Detection Kit S7101; Chemicon, Temecula, CA). For fixed, paraffin-embedded sections from female rodent mammary gland each rabbit eye, the TUNEL assay was performed on the first section of retrieved 3 to 5 days after the weaning of rat pups. Sections were each hemi-block of tissue. Sections were cut at 5 ␮m, deparaffinized, observed with a photomicroscope (BX-50; Olympus) and the immuno- and rehydrated through graded concentrations of alcohol. Protein was labeled cells were identified on each section. then digested by treating the tissue slides with proteinase K (00-3011; Zymed, South San Francisco, CA) at room temperature for 15 minutes. Statistical Analysis The slides were rinsed in distilled water, followed by the quenching of Statistical analysis was performed with commercial software (SPSS for endogenous peroxidase activity with 3% hydrogen peroxide for 5 Windows, ver. 13.0; SPSS, Chicago, IL). An independent, two-sample minutes. The apoptosis detection processing included the application test (Mann-Whitney U test) was used to calculate differences in the of an equilibration buffer directly to each specimen, followed by 15 to mean percentage of apoptotic TUNEL-positive cells between the con- 20 seconds’ incubation. Then, 30 ␮L of working strength deoxynu- trol and any study group or between two study groups. Data in the cleotidyl transferase (TdT enzyme) was applied directly to each spec- figures are expressed as the mean Ϯ SD. P Ͻ 0.05 was considered imen, after which the tissue slides were incubated in a humidified statistically significant. chamber at 37°C for 1 hour. Each specimen was put in a coplin jar containing prewarmed, working-strength stop-wash buffer and incubated for 10 minutes and antidigoxigenin peroxidase was applied to RESULTS each specimen. The specimens were incubated in a humidified chamber for 30 minutes at room temperature. Finally, diaminobenzidine Bevacizumab and pegaptanib sodium were tolerated well. No (DAB) was used as the color substrate. Sections were counterstained apparent changes in the retinas were noticed by fundus exam- with 0.5% methyl green and mounted. ination in all groups at the different times. No conjunctival Formaldehyde fixed, paraffin-embedded sections from female ro- hyperemia, anterior chamber reaction, or any pupillary abnor- dent mammary gland retrieved 3 to 5 days after the weaning of rat pups mality was seen. The vitreous demonstrated no opacities or were obtained from the manufacturer and used as the positive control. degeneration. The negative control experiments were performed without active TdT All study and control eyes were evaluated by H&E staining. but including proteinase K digestion to control for nonspecific incor- At all time points after intravitreal injection of bevacizumab or poration of nucleotides or for nonspecific binding of enzyme conju- pegaptanib sodium, the overall appearance of the retina did gate. not differ significantly from the control group. The morphologic appearances of the cornea, iris, retina, choroid, sclera, Quantitative Analysis of TUNEL. For each sample, the num- and optic nerve were normal in both control and study groups. ber of TUNEL-positive cells in the outer nuclear layer (ONL) was No sign of retinal necrosis, cystic degeneration, or hypocellu- determined in three meridian sections through the optic nerve head. larity of the nuclear layer was observed in any of the groups. Sections were examined at high magnification, and color images were Some polymorphonuclear leukocytes were noted in all groups, obtained with a digital camera (DP-71 CCD; Olympus). Apoptotic cells suggesting inflammatory reaction in the limited areas of injec- were counted in three different microscopic fields of each retinal tion site at the limbus. section (NIS-Elements BR 2.30; Image Analysis System; Nikon, Tokyo, All saline-injected and control eyes showed caspase-3- and Japan). The number of apoptotic, TUNEL-positive cells is expressed as -9-positive cytoplasmic staining in the inner and outer limiting the ratio of TUNEL-labeled nuclei to the total number of nuclei exam- membrane, and inner plexiform layer (Fig. 1B). A similar pat- ined. The mean of the three sections was used as independent data for tern of staining was observed in eyes in which bevacizumab or each eye. pegaptanib sodium had been injected from both the 14-day and Immunohistochemical Staining. Immunohistochemical 3-month groups (Figs. 1C–H). In the study groups, immunohis- staining for caspase-3 and -9 was performed by the standard streptavi- tochemical staining was also demonstrated in the outer plexi- din-biotin-peroxidase method (cat no. 85-6743, Histostain Plus Rabbit form and photoreceptor cell layers. Primary Kit; Zymed). Tissue sections were deparaffinized with xylol TUNEL-positive staining was observed in the ganglion cell and rehydrated with graded alcohol concentrations and then subse- layer, inner nuclear layer, and retinal pigment epithelium in quently in distilled water. To obtain better and more specific staining, both early and late-term groups into which bevacizumab or the antigen retrieval method was used as follows: Sections were put in pegaptanib sodium had been injected. A similar pattern of a coplin jar containing citrate buffer (pH 6.0; (15-M103; Bio Optica, labeling was also observed in eyes in the saline-injection and Milan, Italy) and irradiated in microwave oven for 4 ϫ 2 minutes at a control groups (Fig. 2). TUNEL staining detected apoptotic power of 800 W. Then, the sections were incubated in the same buffer cells in the ONL only in the study groups (Figs. 2C–J). In- solution for 15 minutes. The specimens were washed in 0.01 M creased apoptotic staining in the ONL was more evident in phosphate-buffered saline (pH 7.4)ϩTween 20 (PBST, 51966; Dako, groups 3, 4 (Fig. 2D), and 6 (Figs. 2E, 2F; 1.25-mg day 14 group, Carpinteria, CA) solution (0.1% solution). After the antigen retrieval, 2.5-mg day 14 group, and 0.625-mg three-injection group). endogenous peroxidase activity was quenched by 3% hydrogen per- Increased apoptosis in photoreceptor cell nuclei was seen at oxide for 10 minutes, and the sections were exposed to blocking similar levels in all groups that received pegaptanib sodium serum for 60 minutes to block any nonspecific reaction sites. Caspase-3 (Figs. 2G–J). The levels of apoptotic, TUNEL-positive cells are (sc-7148, diluted 1:50; rabbit polyclonal antibody; Santa Cruz Biotech- displayed in Figures 3 to 5. nology, Santa Cruz, CA) and caspase -9 (rabbit polyclonal antibody, Apoptotic activity, shown by positive TUNEL staining, was sc-8355, diluted 1:50; Santa Cruz) were then added and the sections analyzed statistically and seen to increase toward higher doses were incubated at ϩ4°C overnight. Antibodies were diluted with an in eyes receiving bevacizumab (Figs. 3, 4). In the bevacizumab- antibody diluent solution (cat no. 00-3118). The specimens were injected groups, 0.625-, 1.25-, and 2.5-mg doses at 14 days and rinsed with 0.01 M PBS three times. Tissue sections were then treated 0.625 mg at 3 months (three injections over 3 months) caused with biotin-labeled rabbit anti-goat immunoglobulin G at room temper- significantly more apoptotic activity when compared to the ature for 15 minutes, rinsed with PBST, followed by adding streptavi- 0.25-mg dose at day 14 (mean percentage of apoptotic cell din-biotin-peroxidase complex at room temperature for 15 minutes. counts: 43.0 Ϯ 2.2, 55.3 Ϯ 2.6, 64.5 Ϯ 4.7, 49.8 Ϯ 4.3, and

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FIGURE 1. Caspase-3 immunostaining in all groups. (A) Negative control. (B) Antibody labeling was detected along the inner plexiform layer (IPL) and the inner and outer limiting membranes (ILM/OLM) in the saline-injection groups. (C–E) Caspase-3–positive immunostaining in the bevacizumab group. A staining pattern similar to that in the saline group was observed in the IPL. How- ever, positive staining was detected in the outer plexiform layer (OPL) and photoreceptor cell layer (PCL). No differences in staining intensity were detected between the groups. (F–H) Caspase-3–positive staining in the pegaptanib sodium group. Anti- body labeling was observed along the ILM, OLM, IPL, OPL, and PCL in retina. Bev, bevacizumab; Peg, pegaptanib sodium.

32.0 Ϯ 1.8; P ϭ 0.02 for all comparisons). When compared to ity between a single injection of 0.625 mg bevacizumab and the saline-injection group, a single injection of 0.25 mg bevaci- three injections over three months (P ϭ 0.02). zumab at day 14 did not cause significant apoptosis (P ϭ 0.66); The lowest dose of 0.15 mg pegaptanib sodium at day 14, 0.625, 1.25, and 2.5 mg bevacizumab at day 14 caused signif- which corresponds to the dose used clinically in humans, as icantly more apoptotic activity when compared with that in well as doses of 0.3 and 0.6 mg pegaptanib sodium at day 14 the saline-injection group (Pϭ 0.03, 0.02, and 0.02, respec- caused significantly more apoptotic activity than saline (45.3% tively). A single dose of 0.625 mg bevacizumab evaluated at 14 Ϯ 6.1%, 49.3% Ϯ 4.6%, 48.5% Ϯ 2.6%, and 32.3% Ϯ 3.1%, days or at 3 months after the injection and at 3 months after respectively; P ϭ 0.02 for three doses of pegaptanib compared three injections (one per month) of caused more apoptotic to saline; Figs. 3, 5). A single injection and three injections over activity than the saline injection (P ϭ 0.03, 0.02, and 0.02, 3 months of 0.15 mg pegaptanib sodium (45.8% Ϯ 5.7% and respectively). The difference in apoptotic activity between the 44.3% Ϯ 4.0%; percentage of mean apoptotic cells, respec- 0.625- and 1.25-mg doses of bevacizumab or between the 1.25- tively) also caused significant apoptotic activity when com- and 2.5-mg doses was statistically significant (P ϭ 0.02). There pared to that in the saline-injection group (P ϭ 0.02 for each was also a statistically significant difference in apoptotic activ- comparison). When the pegaptanib sodium groups were com-

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FIGURE 2. Detection of apoptotic cells by TUNEL staining. (A) Sham control. (B) Positive staining was observed in ganglion cell layer (GCL) and inner nuclear layer (INL) in the saline-injection group. (C–F) Apopto- sis in the bevacizumab groups. Apo- ptosis was seen in the GCL, INL and ONL. An evident increase in apoptotic staining in ONL was seen in groups 4 and 6 (2.5 mg /day14 and 0.625 mg/ 3-injection groups). (G–J) Apoptosis in the pegaptanib sodium group. TUNEL-positive staining was detected in GCL, INL, and ONL. (F, J) TUNELϩmethyl green staining in groups 6 and 11. Bev, bevacizumab; Peg, pegaptanib sodium.

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FIGURE 5. Comparison of the percentage of apoptotic cells (mean Ϯ FIGURE 3. The distribution of apoptotic cells in the ONL for all groups SD) in the ONL of the control and saline groups with the pegaptanib (median cell count in box-and-whisker format). Bevacizumab-treated sodium-injected groups. G7: 0.15 mg /day 14; G8: 0.3 mg/day 14; G9: groups: 1: 0.25 mg /day14; 2: 0.625 mg/day 14; 3: 1.25 mg/day14; 4: 0.6 mg/day 14; G10: 0.15/one injection, 90 day; and G11: 0.15 mg/ 2.5 mg/day14; 5: 0.625/one injection, 90 days; and 6: 0.625 mg/three three-injection, 90 days. *Statistically significant when compared to the injections, 90 days. Pegaptanib sodium treated groups: 7: 0.15 mg/ saline-injection group (P Յ 0.05). day14; 8: 0.3 mg/day 14; 9: 0.6 mg/day 14; 10: 0.15/one injection, 90 days; and 11: 0.15 mg/3 injections, 90 days; group 12: saline control; and group 13: sham control. pegaptanib; P ϭ 0.88 and 0.08, respectively; Fig. 3). However, apoptotic activity between fourfold clinical doses of both drugs (2.5 mg bevacizumab versus 0.6 mg pegaptanib) was pared to each other, no significant difference in apoptotic significantly different (P ϭ 0.02). In the long-term groups, activity was found between the different dose groups in con- pegaptanib sodium and bevacizumab did not cause signifi- trast to that in the bevacizumab groups. Higher concentrations cantly different apoptotic activity in clinical doses when com- of pegaptanib sodium did not cause more apoptotic activity pared to each other (P ϭ 0.88 and P ϭ 0.15 for single and three than did lower concentrations. injections, respectively). When the pegaptanib sodium groups were compared to the bevacizumab groups, no significant difference in apoptotic activity was observed between the clinical doses (0.625 mg bevacizumab versus 0.15 mg pegaptanib) and between the DISCUSSION twofold clinical doses (1.25 mg bevacizumab versus 0.3 mg The purpose of our study was to reveal any toxic side effects of bevacizumab and pegaptanib sodium after intravitreal injection, and we evaluated possible apoptosis in photoreceptor cells for different doses of tested agents. Overall, no toxicity- related adverse effects were observed by clinical and routine light microscopic assessment (data not shown), and no inflammatory reaction was demonstrated in the experimental groups. In our previous study, we reported that intravitreal bevacizumab injection was related to mitochondrial disruption in the inner segments of photoreceptors documented by electron microscopy and also increased apoptosis in inner layers of the retina by caspase-3, caspase-9, and bax/bcl-2 immunostaining, but we did not perform quantitative evaluation of apoptosis in the previous study.13 In the present study, we used the TUNEL method for detection of DNA fragmentation in nuclei of apoptotic cells in nuclear layers. Caspase-3 and -9 staining is a marker of apoptosis that is based on cytoplasmic localization in cells containing activated caspase and shows good correlation with morphologic apoptosis demonstrated by the TUNEL method.23 Statistical analysis was performed for the sections stained with the TUNEL method. In our study, we also detected apoptosis in the control eyes. It seems that the apoptotic process continues physiologically FIGURE 4. Comparison of the percentage of apoptotic cells (mean Ϯ SD) in the ONL of the control and saline-injection groups with the during life. Although there is no study in which the apoptotic bevacizumab-injected groups. G1: 0.25 mg/day 14; G2: 0.625 mg/day process was specifically evaluated by the TUNEL method in 14; G3: 1.25 mg/day 14; G4: 2.5 mg /day 14; G5: 0.625/one injection, normal rabbit retina, it has been demonstrated in retinal cells in 11,24 90 days; and G6: 0.625 mg/three injections, 90 days. *Statistically the control eyes of several experimental animal studies. significant when compared to the saline-injection group (P Յ 0.05). Apoptotic activity was shown in ganglion cells and inner and

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outer nuclear cell layers in the control groups of the porcine cells as well as Mu¨ller cells require VEGF as a survival factor retina-pigment epithelium-choroid culture.11 the apoptotic under normal conditions. Dependence on VEGF signaling may process was also shown in normal primate photoreceptor cells be due to the high metabolic rates of retinal photoreceptor with lower levels.24 In our previous study we detected apo- cells and their extreme sensitivity to hypoxia.33 Therefore, in ptosis in control rabbit eyes with caspase and bax/bcl-2 immu- situations especially where the retina is under threat of hyp- nostaining.13 Whether nonspecific background staining in- oxia, a complete deficiency of the physiological level of endog- creased the apoptotic cell count in the sham and saline control enous VEGF may be critical for photoreceptor cells. eyes remains to be answered in future studies. In a mouse model, systemic neutralization of VEGF led to Our finding that bevacizumab caused apoptosis at higher significant cell death in the inner and outer nuclear cell layer at doses may suggest that increasing the dosage with intravitreal 14 days as well as loss of visual function.34 Cell death was more bevacizumab can cause greater toxic effects on retinal neuro- evident in the ONL where a high number of photoreceptor nal cells. Similar dose-related adverse effects were not found cells are present. It has been suggested that the physical asso- for pegaptanib sodium. Pegaptanib sodium–related apoptosis ciation between the photoreceptors that express VEGF recep- was similar among the clinical, twofold, and fourfold dose tors and the Mu¨ller cells that express VEGF points to a para- groups. This dose-independent effect remains to be investi- crine interaction in which Mu¨ller cells provide VEGF as a gated. When the apoptotic activity of bevacizumab and pe- neurotrophic signal to photoreceptors. VEGF blockage has also gaptanib was compared, it was seen that there was no differ- been demonstrated to cause ganglion cell apoptosis in a rat ence in the toxicity of these agents at clinical and twofold model.35 doses, but bevacizumab caused more evident toxicity in com- The neuroprotective effect of VEGF has been suggested to parison to pegaptanib when fourfold doses of both agents involve suppression of cell-death pathways mediated by were used. After pegaptanib injection, other VEGF isoforms caspase-3, which plays a key role in apoptosis and is induced may protect the retina when only the 165 isoform was by caspase 9. Evidence suggests that VEGF exerts its antiapo- blocked. Another explanation may be a faster clearance of ptotic action through VEGFR-2 receptors and the phosphati- pegaptanib sodium from the vitreous. However, pegaptanib dylinositol 3Ј-kinase/Akt signal transduction system. The sodium in clinical doses was also seen to evoke apoptosis when serine-threonine protein kinase Akt contributes to the inhibi- compared with the saline control. The other finding from the tion of apoptosis by activating antiapoptotic proteins through present study was that the commonly used clinical dose of the transcription factor NF␬B and inhibiting proapoptotic sig- bevacizumab corresponding to 0.625 mg in rabbit eyes caused naling by Bad, caspase-9, and other effectors. Akt inhibits the a significant increase in apoptosis in comparison to the saline proapoptotic Bcl-2 family member Bad and caspase-9. The injection. In addition, repeated injections of the same dose of proapoptotic protein Bad acts at the surface of the mitochon- bevacizumab were related to more apoptosis than was a single drial membrane to decrease the mitochondrial transmembrane dose. Thus, in clinical practice it should be borne in mind that potential and promotes leakage of cytochrome c. Released bevacizumab should be used in the lowest possible doses. cytochrome c complexes with and activates Apaf-1, which Anti-VEGF activities of bevacizumab and pegaptanib to- binds to procaspase-9, and converts this enzyme into its pro- gether with ranibizumab have been compared in vitro. Pe- teolytically active form, caspase-9. Caspase-9 leads to apoptosis gaptanib showed no effect, whereas the others neutralized through nuclear damage (DNA fragmentation, DNA muta- VEGF completely at clinical doses.10 Pegaptanib sodium has tions). The inhibition of Bad and caspase-9 by Akt results in been verified to be effective in patients with ARMD with proteolytic cascade blockage and prevents caspase-3-mediated multicenter clinical trials, but the results in these trials re- DNA fragmentation. However, further studies are needed to mained in the shadow of results of trials of complete VEGF delineate the additional mechanisms by which VEGF exerts its inhibitors. When considering the VEGF-related physiological antiapoptotic effects on retinal neuronal cells.36 functions and possible toxic effects of pan-VEGF antagonism, Our results regarding the effect of anti-VEGF therapy on pegaptanib sodium was regarded as more reliable for both apoptosis are supported by those in previous studies. In- ocular and systemic safety. It is not presently clear whether creased apoptotic cell death detected at inner and outer pho- related results from in vitro or experimental animal studies, toreceptor layers in our study is consistent with the findings including the present study might help to explain clinical that bevacizumab immunoreactivity can be found in several experience with pegaptanib sodium. retinal cell types 14 days after injection, the most prominent of Several reports have demonstrated that VEGF not only in- them being rod photoreceptor outer segments and endothelial duces angiogenesis, but also works as a survival factor for cells of blood vessels.5 tumor and endothelial cells, protecting from apoptosis.25 With- An electron microscopy study showed that intravitreal be- drawal of VEGF leads to endothelial cell apoptosis in vitro and vacizumab caused ultrastructural changes in the choriocapil- in vivo.26 Additional VEGF functions under investigation in- laris with significant reduction of choriocapillaris endothelial clude retinal leukostasis and neuroprotection.27,28 It has been cell fenestration in primate eyes. Atrophy of choriocapillaris or reported that VEGF, besides its neuroprotective effects, has a loss of its endothelial cell fenestrations impairs nutritional neurotrophic influence, by stimulating axonal outgrowth from support, which may lead to functional and morphologic dam- dorsal root ganglia or superior cervical ganglia in culture.29,30 age to the RPE and photoreceptors, with particular adverse In the adult retina, VEGF is expressed by retinal pigment effects if the macular region is affected.14 In addition, in a epithelial cells as well as by neuronal and glial cells in the recent study in which a retinal neovascularization model was ganglion cell layer and inner nuclear cell layer.31 VEGF recep- developed, it has been suggested that caution may be war- tor (VEGFR)-2 is expressed by adult photoreceptor cells and ranted in the treatment of patients with acute or severe retinal Mu¨ller cells. It has been suggested that VEGF and their recep- neovascularization by anti-VEGF drugs such as bevacizumab to tors provide an endogenous mechanism that may protect the prevent capillary nonperfusion and macular ischemia.6 The retina against damage of different origins and may also promote established neuroprotective and neurotrophic effect of VEGF axonal regeneration in central nervous system (CNS) lesions.29 may explain an increase in apoptosis in retinal neuronal cells It has also been suggested that tissue inhibitor of matrix met- by intravitreal anti-VEGF injections. alloproteinase-1 and VEGF in combination promote the sur- Bevacizumab, ranibizumab, and pegaptanib significantly vival of photoreceptor cells in rat retinal explants, possibly by suppressed choroidal endothelial cell proliferation in vitro affecting a caspase-3 signaling pathway.32 Retinal neuronal when used at the currently recommended intravitreal dose.

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However, when used at the established dose, none of the drugs 4. Leung DW, Cachianes G, Kuang WJ, et al. Vascular endothelial was superior over the others with respect to endothelial cell growth factor is a secreted angiogenic mitogen. Science. 1989;246: growth inhibition. No cytotoxicity was detectable for pe- 1306–1309. gaptanib and bevacizumab at the respective concentrations 5. Heiduschka P, Fietz H, Hofmeister S, et al. Penetration of Bevaci- 12 zumab through the retina after intravitreal injection in the monkey. that included the clinical dose for both drugs. In another Invest Ophthalmol Vis Sci. 2007;48:2814–2823. study, it was suggested that bevacizumab, administered at or 6. Ameri H, Chader GJ, Kim JG, et al. The effects of intravitreous above the dose normally used in clinical practice, is safe in the bevacizumab on retinal neovascular membrane and capillaries in short term for retina pigment epithelium (RPE) cells, rat retinal rabbits. Invest Ophthalmol Vis Sci. 2007;48:5708–5714. neurosensory cells, and human microvascular cells in vitro.37 7. Dib E, Maria M, Longo-Maugeri IM, et al. Subretinal bevacizumab However, our results suggest that intravitreal administration of detection after intravitreous injection in rabbits. Invest Ophthal- high doses of anti-VEGF agents does matter, especially with mol Vis Sci. 2008;49:1097–1100. bevacizumab, when safety is evaluated in regard to apoptosis. 8. Shahar J, Avery RL, Heilweil G, et al. Electrophysiologic and retinal penetration studies following intravitreal injection of bevacizumab When apoptosis in the retina was evaluated in porcine retina- (Avastin). Retina. 2006;26:262–269. RPE-choroid cultures exposed to bevacizumab (concentrations 9. Manzano RP, Peyman GA, Khan P, et al. Testing intravitreal toxicity up to five times higher than those used clinically) for 3 days, no of bevacizumab (Avastin). Retina. 2006;26:257–261. increase in apoptotic cells in the ganglion cell layer or RPE- 10. Klettner AK, Roider J. Comparison of bevacizumab, ranibizumab choroid was observed by TUNEL labeling.11 In contrast to and pegaptanib in vitro efficiency and possible additional path- these findings, our results showed increased apoptosis at ways. Invest Ophthalmol Vis Sci. 2008;49:4523–4527. higher doses (especially at a fourfold higher dose than that 11. Kaempf S, Johnen S, Salz AK, et al. Effects of bevacizumab (Avas- used clinically) in an experimental clinical rabbit study. tin) on retinal cells in organotypic culture. Invest Ophthalmol Vis Sci. 2008;49:3164–3170. On the other hand, no toxic effects of intravitreal bevaci- 12. Spitzer MS, Yoeruek E, Sierra A, et al. Comparative antiproliferative zumab on mouse retina, as examined by histologic and TUNEL and cytotoxic profile of bevacizumab (Avastin), pegaptanib (Macu- apoptosis analysis, were found even at high doses in a study gen) and ranibizumab (Lucentis) on different ocular cells. Graefes published very recently.38 Although the results from this study Arch Clin Exp Ophthalmol. 2007;245:1837–1842. are contrary to our results, differences in species may compro- 13. Inan UU, Avci B, Kusbeci T, et al. Preclinical safety evaluation of mise interpretation for comparison of conflicting results. This intravitreal injection of full-length humanized vascular endothelial monoclonal antibody has been shown to inhibit tumor growth growth factor antibody in rabbit eyes. Invest Ophthalmol Vis Sci. in a dose-dependent manner in various animal models.39 Bev- 2007;48:1773–1781. 14. Peters S, Heiduschka P, Julien S, et al. 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