ABSTRACT
Objectives:
To assess the effects of anti-vascular endothelial growth factor (VEGF) drugs on retinal pigment epithelium cell culture.
Materials and Methods:
Aflibercept (0.5 mg/mL), bevacizumab (0.3125 mg/mL), and ranibizumab (0.125 mg/mL) were applied to retinal pigment epithelium cell cultures isolated from the enucleated eyes of New Zealand white rabbits. Viability, apoptosis, proliferation, and senescence of the cells were evaluated in control and drug-treated cultures at the end of 72 hours.
Results:
Cells treated with aflibercept showed increased viability and decreased apoptosis compared to the control culture and both the bevacizumab- and ranibizumab-treated groups (p<0.05). Statistically increased apoptosis and decreased viability were found in the bevacizumab and ranibizumab-treated groups compared with the control group (p<0.05). There were no statistically significant differences in cell proliferation and senescence between the groups (p>0.05).
Conclusion:
Anti-VEGF drugs did not affect senescence or proliferation of retinal pigment epithelium cells. Aflibercept was found to decrease apoptosis and increase cell viability, while ranibizumab and bevacizumab increased apoptosis and reduced cell viability in retinal pigment epithelium culture.
Introduction
It is well known that vascular endothelial growth factor A (VEGF-A) is a main mediator of angiogenesis and increased vascular permeability in retinal vascular disorders.1,2,3,4 The inhibition of vascular endothelial growth factor (VEGF) has been a key point in experimental and clinical studies under research. The effectiveness of intravitreal administration of various anti-VEGF agents is well established in the treatment of macular edema of different origins.5 The mechanism of action of these drugs when delivered intravitreally is complex and involves the blocking of various types of VEGFs, decreased permeability of newly formed blood vessel walls, and reduced swelling of the retinal layers. In recent years, several reports have demonstrated the impact of anti-VEGF drugs upon different cell cultures in vitro.6,7,8,9,10,11 Our goal was to investigate the effects of anti-VEGF drugs on viability, apoptosis, proliferation, and senescence in retinal pigment epithelium (RPE) cell culture, which can serve as an in vitro model.
In this study, we compared the proliferative and cytotoxic effects of aflibercept (0.5 mg/mL), bevacizumab (0.3125 mg/mL), and ranibizumab (0.125 mg/mL) on RPE cell cultures by evaluating viability, apoptosis, proliferation, and senescence in control and drug-treated cells after 72 hours.
Materials and Methods
Results
Discussion
This is the first study in the literature to assess the senescence effects of anti-VEGF drugs and to identify that these agents do not seem to have a significant effect on the senescence of RPE cells in vitro. In addition, we have showed that RPE morphology and proliferation are also not affected by the anti-VEGF drugs most commonly used in retinal diseases. However, aflibercept increased viability and decreased apoptosis, while bevacizumab and ranibizumab had the opposite effect.
Intravitreal injection has become a widely used delivery route of various therapeutic agents for the treatment of vasoproliferative ocular diseases.15 Current anti-VEGF therapies delivered intravitreally include ranibizumab and aflibercept, as well as off-label bevacizumab. A main functional difference between aflibercept and other anti-VEGF agents is blockage of VEGF-B, PGF1, and PGF-2 in addition to VEGF-A isoforms. To best of our knowledge there are limited in vitro studies in the literature evaluating the safety and efficacy of ranibizumab, aflibercept, and bevacizumab. In the present study, aflibercept was found to decrease apoptosis and increase cell viability. In contrast, ranibizumab and bevacizumab were observed to increase apoptosis and reduce cell viability in RPE cultures. Retinal pigment epithelium-derived VEGF is a vital mediator for support of the choriocapillaris. Several clinical studies have shown a correlation between subfoveal choroidal thickness and RPE atrophy progression, which suggested that decreased blood supply might promote RPE atrophy during anti-VEGF treatment.16,17 We also noticed that most of the experimental studies on the adverse effects of the anti-VEGF drugs were carried out on RPE cell linings with intact choriocapillaris. Our study is performed on an isolated RPE cell line and may be able to show the direct toxic effects of anti-VEGF drugs on RPE. In a study performed on newborn rabbits, Cam et al.18 showed that at 3 weeks after injection, all anti-VEGF drugs caused low levels of serum anti-VEGF concentrations and induced apoptosis as determined with apoptotic index, which was described as the percentage of apoptotic TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) positive cells of tissues. Malik et al.19 also studied the safety profiles of various concentrations of anti-VEGF drugs on human RPE cells. They reported that while ranibizumab and aflibercept did not cause mitochondrial toxicity or cell death, bevacizumab and aflibercept revealed mild mitochondrial toxicity, though they also did not cause significant cell death at clinical doses. However, in our study all of the drugs caused a significant difference in cell viability. Malik et al.19 treated the culture media with anti-VEGF drugs at concentrations they considered to be the clinical dose, assuming the amount of intravitreal injected drug spreads equally throughout the 4 mL human vitreous and modifying the doses accordingly. No such modification was performed in our study, which is a limitation of our study and could explain our conflicting results.
In our study, anti-VEGF drugs had no effect on the senescence or proliferation of RPE cells at after 72 hours. Cellular senescence is a program activated by normal cells in reaction to various stress factors such as oxidative stress, DNA damage, oncogene activity, and inadequate culturing conditions.20,21 Conventionally, when cells enter senescence they exhibit substantial morphological changes. The cells spread out and flatten, which is usually followed by increasing SA β-galactosidase activity.14 To best of our knowledge there are no studies evaluating the effects of anti-VEGF drugs on senescence in the current literature. Our data showed that none of the anti-VEGF drugs affected senescence in RPE cells at the end of the 72 hours. Recently, Zhuge et al.22 reported that fullerenol, an effective free radical scavenger and antioxidant, could salvage RPE cells from oxidative stress-induced senescence due to its antioxidant effect. They suggested that the protective effect of fullerenol is crucial for the development of new treatment strategies in oxidative stress-associated retinal disorders such as age-related macular degeneration (AMD). In addition, Kernt et al.23 investigated the antiapoptotic and cytoprotective effects of idebenone, a benzoquinone derivative that is structurally related to ubiquinone (coenzyme Q10), on optic nerve head astrocytes (ONHA) under oxidative stress. They concluded that idebenone reduced senescence, oxidative stress, and apoptotic cell death in cultured ONHA in vitro. One of the limitations of our study is that we studied the effects of drugs in healthy retinas, not under the drugs’ indicated disease states such as AMD, which would have already had oxidative stress and changes in RPE senescence. Therefore, we do not know the senescence effects of the drugs in already oxidative stress-induced conditions.
Recently, Spitzer et al.8 compared the antiproliferative and cytotoxic effects of bevacizumab, pegaptanib, and ranibizumab on different ocular cells. When applied to choroidal epithelial cells, they observed reductions in cell proliferation of 44.1% with ranibizumab versus 38.2% and 35.1% with bevacizumab and pegaptanib, respectively, although the difference between them was not statistically significant. They reported that bevacizumab, pegaptanib, and ranibizumab significantly suppressed choroidal endothelial cell proliferation and concluded that when used at the currently established doses, none of the drugs was superior over the others in respect to endothelial cell growth inhibition. Our study showed no difference in the proliferation rate of the RPE line in the treated groups compared to controls.
Another study compared the effects of ranibizumab, pegaptanib, and bevacizumab at intravitreal dose range on human umbilical vein endothelial cells (HUVEC).10 The results indicated that ranibizumab and bevacizumab significantly increased apoptosis of HUVEC, similar to our results in RPE cells. Clinically applied doses of these drugs, but not pegaptanib, caused significantly reduced cellular proliferation without causing cytotoxic effects at all concentrations used. In addition, the active form of VEGF receptor-2 expression was decreased relative to controls after incubation with bevacizumab (to 66% of control values), ranibizumab (78%), and pegaptanib (86%).
Schnichels et al.11 investigated the cytotoxicity and antiproliferative activity of aflibercept, bevacizumab, and ranibizumab on different ocular cells (ARPE19, RGC-5, and 661W) and concluded that aflibercept does not cause changes in cell morphology, induce apoptosis, or permanently decrease cell viability, cell density, or proliferation in any cell line or concentration investigated. In addition, aflibercept slightly upregulated or downregulated certain VEGF-related factors, but the changes were not significant when compared to bevacizumab and ranibizumab. The ARPE19 cell line was derived from human RPE in their study, whereas ours was derived from rabbits. VEGF-TrapR1R2 (aflibercept) is composed of entirely human sequences and was constructed to bind human VEGF isoforms.24 Holash et al.24 stated that despite its wholly human nature, VEGF-TrapR1R2 binds all species of VEGF tested, from human to chicken VEGF, yet in their study the experiments were shown on mouse, rat, and humans. The conflicting results obtained in our study and that of Schnichels et al.11 may be attributable to different responses shown by the cell lines of different origin used in the studies.
It is known that most eyes with AMD require long-term anti-VEGF treatment, and it is the constant neutralization of VEGF, which is crucial for ocular homeostasis, that may lead to adverse effects.25
Study Limitations
One of the limitations of our study is that anti-VEGF drugs are used repeatedly, most commonly administered as 3 monthly loading doses followed by repeated injections monthly or pro re nata (as needed) thereafter. In contrast, our study represents results after a single injection, and longitudinal studies showing the effects of more and repeated injections as in real life should be planned.
Çalışmanın Kısıtlılıkları
Çalışmamızın kısıtlamalarından biri, anti-VEGF ilaçların en sık olarak 3 aylık yükleme dozunu takiben aylık tekrarlayan enjeksiyonlar yapılması ya da gerektikçe uygulanması gibi tekrar tekrar kullanılmasıdır. Aksine, çalışmamız sadece bir enjeksiyondan sonra ortaya çıkacak sonuçları temsil etmektedir ve gerçek hayatta olduğu gibi tekrarlanan enjeksiyonların etkilerini gösteren boylamsal çalışmalara ihtiyaç vardır.
Conclusion
In conclusion, our study reveals that anti-VEGF drugs had no effects on senescence and proliferation of RPE cells. Aflibercept was found to decrease apoptosis and increase cell viability. In contrast, ranibizumab and bevacizumab were observed to increase apoptosis and reduce RPE cell viability. In the literature, there are no studies evaluating the effects of anti-VEGF drugs on senescence. We believe that our study will guide future research in this respect and experimental and preclinical studies will be needed to confirm our in vitro findings.
