An In-vitro Evaluation of a New Approach in AMD: Effects of the Combination of Resveratrol and Anti- VEGFs on ARPE-19 Cells

An In-vitro Evaluation of a New Approach in AMD: Effects of the Combination of Resveratrol and Anti- VEGFs on ARPE-19 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An In-vitro Evaluation of a New Approach in AMD: Effects of the Combination of Resveratrol and Anti- VEGFs on ARPE-19 Cells Onur KONUKCU, Mehmet ARGUN, Ömer ÇELİK, Özlem TÖK, Levent TÖK, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4790772/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to determine how resveratrol combination separately with the anti-VEGF agents ranibizumab, aflibercept and ziv-aflibercept affects ARPE-19 cells in vitro. The cells were assigned to twelve groups as follows: Control, Cobalt Chloride (Cob), Resveratrol (RSV), Ranibizumab (RNZ), Aflibercept (AFL), Ziv-aflibercept (ZFL), RNZ + RSV, AFL + RSV, ZFL + RSV, RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob. The Control group was incubated for 48 hours with no treatment, while the remaining groups received RSV, RNZ, AFL or ZFL (alone or in combination) for 24 hours and then the cells in the relevant groups were exposed to CoCl 2 for 24 hours more. Mitochondrial reactive oxygen species (MitROS), cytosolic reactive oxygen species (CytROS), mitochondrial membrane depolarization (MitDep), caspase-3, caspase-8, caspase-9, cell viability, apoptosis and VEGF-A levels were assessed by confocal microscopy, plate reader and ELISA techniques. Resveratrol, alone or in combination with anti-VEGF’s, significantly decreased the levels of MitROS, MitDep, CytROS, caspase-3, caspase-8 and caspase-9 (p < 0.001). Resveratrol also increased cell viability and decreased apoptosis and VEGF-A levels (p < 0.001). According to our findings, combining resveratrol with anti-VEGFs may have a beneficial therapeutic effect on the treatment of AMD. Aflibercept AMD Ranibizumab Resveratrol Ziv-aflibercept Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Age-related macular degeneration (AMD) is a chronic condition that leads to loss of central vision in the elderly individuals [ 1 ]. AMD is the most common cause of blindness in developed countries for people older than 60 years, accounting for 8.7% of all blindness cases globally and is projected to impact over 280 million people by 2040 [ 2 ]. Anti-VEGF agents are the most successful treatment for AMD, as a VEGF-A is essential for the formation and growth of choroidal neovascularization, which is a major cause of vision loss in neovascular AMD [ 3 ]. The agents used for this purpose are ranibizumab, aflibercept and bevacizumab [ 4 ]. Ziv-aflibercept, which is not approved for eye use, has been used more often because of its cost-effectiveness [ 5 ]. Macular atrophy is associated with vision loss in patients with AMD [ 6 ]. The macular atrophy progresses even when patients receive anti-VEGF therapy. The cause of macular atrophy progression is unknown. Oxidative stress and apoptosis may contribute to macular cell death, which leads to macular atrophy progression [ 7 ]. The combination of anti-VEGFs with some molecules that reduce macular atrophy progression may improve the treatment options for AMD. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic phytoalexin produced from phenyl alanine by stilbene synthase in plants [ 8 ]. It attracted medical interest with the “French Paradox”, which proposes that red wine with high levels of resveratrol is good for cardiovascular health [ 9 ]. Resveratrol affects various physiological processes, such as oxidative stress, inflammation, the cell cycle and proliferation, metastasis, apoptosis, and angiogenesis [ 10 ]. It has a strong antioxidant and anti-inflammatory effect [ 11 – 13 ]. Resveratrol has been studied for its cardioprotective, neuroprotective, chemotherapeutic, and anti-aging effect [ 14 – 17 ]. Resveratrol has shown antiangiogenic, antiplatelet and antiproliferative effects [ 18 – 20 ]. In-vitro studies have demonstrated that resveratrol can protect retinal pigment epithelium cells (RPE) from oxidative stress and apoptosis caused by sodium iodate and autoimmunity [ 21 , 22 ]. Resveratrol has been reported to protect against light-induced retinal degeneration [ 23 ]. It has been shown that it inhibits VEGF secretion induced by hypoxia and inflammatory cytokines such as IFN-γ, TNF-α, IL-1β, and TGF-β [ 24 ]. Therefore, resveratrol may be a potential therapeutic agent for AMD, in which inflammation, cell death-related atrophy and VEGF-based neovascularization play key roles in its pathogenesis. The aim of this study was to examine the effects of adding resveratrol to anti-VEGFs (ranibizumab, aflibercept, ziv-aflibercept) on ARPE-19 cells in-vitro. We measured and analysed intracellular and mitochondrial ROS levels, mitochondrial membrane depolarization, caspase-3, caspase-8 and caspase-9 activities, cell viability, apoptosis, and VEGF-A levels in the anti-VEGF treatment groups with or without resveratrol to determine the effects of resveratrol in combination treatment. Materials and Methods Chemicals and Solutions The human retinal pigment epithelium cell line (ARPE-19) was acquired from American Type Cell Culture (ATCC-Virginia, USA, ATCC Number: CRL-2302). Cell culture medium was obtained from Sigma Aldrich (Missouri, USA). APOPercentageTM assay kit was obtained from Biocolor (Belfast, Northern Ireland). Human Vascular Endothelial Cell Growth Factor A (VEGF-A) ELISA kit was procured from Bioassay Technology Lab (Shanghai, China, Cat. No. E0050Hu). All reagents were of analytical grade. Each experiment was performed at least six times. Cell culture ARPE-19 cells were grown in a concoction medium consisting of 1:1 ratio of Dulbecco’s Modified Eagle Medium and Ham’s F12 medium complemented with 10% fetal bovine serum (Biochrom, Germany) and 1% penicillin-streptomycin mixture (Biochrom, Germany) in accordance with the manufacturer's instructions. Cells were used in passages between 2–12. The cell culture operations were implemented at 37°C in a humidified incubator (Heal Force HF90, Japan) with 5% CO 2 [ 25 ]. The Experimental Design The final volume of growth medium per flask in all groups was determined as 4 ml which is the physiological volume of vitreous and the incubation dose of anti-VEGF drugs was ascertained according to Malik et al.[ 26 ]. The study groups and treatments were followed as: Control: ARPE-19 cells were incubated for 48 h in a standard growth medium and under the conditions described above. Cobalt Chloride (CoCl 2 ) Group (Cob): The cells were incubated for 48 h. In the 24th h of incubation, the cells were treated with CoCl 2 (200 µM) without refreshing the growth medium. Resveratrol Group (RSV): The cells were incubated with RSV (100 µM) for 48 h. Ranibizumab Group (RNZ): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) for 48 h. Aflibercept Group (AFL): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) for 48 h. Ziv-aflibercept Group (ZFL): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) for 48 h. Ranibizumab + Resveratrol Group (RNZ + RSV): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) and RSV (100 µM) for 48 h. Aflibercept + Resveratrol Group (AFL + RSV): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) and RSV (100 µM) for 48 h. Ziv-aflibercept + Resveratrol Group (ZFL + RSV): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) and RSV (100 µM) for 48 h. Ranibizumab + Resveratrol + Cobalt Chloride Group (RNZ + RSV + Cob): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) and RSV (100 µM) for 48 h. In the 24th h of incubation, the cells were treated with CoCl 2 (200 µM) without refreshing the growth medium. Aflibercept + Resveratrol + Cobalt Chloride Group (AFL + RSV + Cob): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) and RSV (100 µM) for 48 h. In the 24th h of incubation, the cells were treated with CoCl 2 (200 µM) without refreshing the growth medium. Ziv-aflibercept + Resveratrol + Cobalt Chloride Group (ZFL + RSV + Cob): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) and RSV (100 µM) for 48 h. In the 24th h of incubation, the cells were treated with CoCl 2 (200 µM) without refreshing the growth medium. Confocal Microscopy Analysis Mitochondrial ROS Generation (MitROS), Mitochondrial Membrane Depolarization (MitDep) and Cytosolic ROS Generation (CytROS) Changes in mitochondrial ROS generation (MitROS), mitochondrial membrane depolarization (MitDep) and cytosolic ROS generation (CytROS) in ARPE-19 cells were observed in an LSCM/800 instrument with a Plan-Apochromat 20×/0.8 objective (Zeiss, Germany). To assess MitROS and CytROS levels; MitoTracker Red CM-H2Xros (MtTrk, 1 µM for 30 min) and DCFH/DA (1 µM for 30 min) probes were applied respectively. The cells were exposed to the fluorescent probe JC-1 (2 µM) for 30 min to determining of MitDep. MtTrk (561 nm), JC-1 (488 nm) and DCFH/DA (488 nm) were activated with a diode argon laser. The ZEN program was used to measure fluorescence scattering as arbitrary unit (a.u.) in the captured red (MtTrk), orange (JC-1) and green (DCFH/DA) images [ 27 , 28 ]. Total Number of Cells and Rate of Cell Death The cell death ratio was assessed in the images obtained after staining with Hoechst 33342 (Thermo Fisher Sci.) and propidium iodide (PI; Thermo Fisher Sci.). Hoechst 33342 (8.1 µM) and PI (1.5 µM) dyes were applied to the dishes with bottom glass. The cells were incubated for 20 min with PI and Hoechst 33342, and then the red and blue images were captured by the LSCM/800. The ratio of PI-positive (dead) cells was calculated by individually counting the number of blue and red cells using the ZEN program [ 29 ]. Plate Analysis Mitochondrial Membrane Depolarization (MitDep) and Cytosolic ROS Generation (CytROS) The oscillations in MitDep and CytROS were measured by an automatic plate reader (Infinite 200 PRO, Tecan Life Sci. Männedorf, Switzerland) with JC-1 and DHR-123 dyes, respectively. The fluorescence scattering density variations of JC-1 and DHR-123 were expressed as a percentage of the control after computing the fluorescence units/mg protein [ 30 , 31 ]. Caspase-3, 8 and 9 The activities of Caspase-3, Caspase-8 and Caspase-9 were detected by using the passive fluorogenic substrates Ac-DEVD-AMC, Ac-IETD-AFC, and Ac-LEHD-AFC (Bachem AG., Bubendorf, Switzerland) respectively. The levels of AMC and AFC which are specifically cleaved by active Caspase-3, Caspase-8 and Caspase-9 were measured by Infinite 200 PRO (Ext: 360–400 nm, Ems: 460–505 nm). The results are expressed as a percentage of the control [ 30 ]. Cell Viability and Apoptosis The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test is a method used to measure cell viability. The principle of the test is that only living cells can reduce a yellow, water-soluble tetrazolium salt to a purple, water-insoluble formazan crystals. Dead cells cannot perform this enzymatic reaction. After drug treatment, the cells were separated and MTT dye was added to the tubes. The cells were incubated in a humidified incubator for 90 min at 37°C. The supernatant was discarded and 150 µL of DMSO was used to solubilize the formazan crystals. An automated multiplate reader (Tecan Infinite 200 Pro) was used to measured the optical densities at 490 and 650 nm (as reference wavelength) and the results were expressed as the fold change relative to control as experiment/control [ 32 , 33 ]. The APOPercentage Apoptosis Kit (Biocolor Ltd., Northern Ireland) was used to assess the level of apoptosis according to a protocol described elsewhere. The results were shown as a percentage of the control [ 30 , 34 ]. VEGF-A Levels VEGF-A levels were determined according to the manufacturer’s instructions by using a commercial ELISA (Cat.No: E0050Hu, BT Lab, China). The curved line had an R 2 of 0.998. The results were given as pg/ml [ 25 , 35 ]. Results Confocal Microscopy Analysis Results The confocal microscopy images and findings are shown in Fig. 1 . The MitROS (Fig. 1 B), MitDep (Fig. 1 C) and CytROS (Fig. 1 D) levels in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups respectively; increased significantly compared to the RSV group (p < 0.001). MitROS, MitDep and CytROS levels in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups, respectively (p < 0.001). The total number of cells (Fig. 1 E) in the RNZ + RSV, AFL + RSV and ZFL + RSV groups increased significantly compared to RNZ, AFL and ZFL groups respectively; decreased significantly compared to RSV group (p < 0.001). The total number of cells in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups increased significantly compared to the Cob, RNZ, AFL and ZFL groups and decreased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups, respectively (p < 0.001). The rate of cell death (Fig. 1 F) in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p < 0.001). The rates of cell death in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups respectively (p < 0.001). Plate Reader Analysis Results The MitDep and CytROS levels measured by a plate reader are shown in Fig. 2 . MitDep (Fig. 2 A) and CytROS (Fig. 2 B) levels in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared with the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p < 0.001). MitDep and CytROS levels in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups respectively (p < 0.001). Caspase-3, Caspase-8 and Caspase-9 activity levels are shown in Fig. 3 . Caspase-3 (Fig. 3 A) and Caspase-9 (Fig. 3 C) activity in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared with the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p < 0.001). Caspase-3 and Caspase-9 activity in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups, there was no statistically significant difference. Caspase-8 (Fig. 3 B) activity increased significantly in the Cob, RNZ, AFL, ZFL, RNZ + RSV, AFL + RSV, ZFL + RSV, RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups compared to the Control and RSV groups; decreased significantly in the RSV group compared to the control group (p < 0.001). There was no significant difference in Caspase-8 activity between the RNZ, AFL, ZFL, RNZ + RSV, AFL + RSV, ZFL + RSV, RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups. Cell viability, apoptosis and VEGF-A levels are presented in Fig. 4 . Cell viability (Fig. 4 A) level in the RNZ + RSV, AFL + RSV and ZFL + RSV groups increased significantly compared to the RNZ, AFL and ZFL groups, respectively (p < 0.001); decreased significantly compared to RSV group. It was determined that cell viability was higher in the groups in which anti-VEGF agents and resveratrol were combined compared to the groups in which anti-VEGF agents were used alone. The cell viability level in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups increased significantly compared to the Cob group; increased significantly compared to the RNZ, AFL and ZFL groups, respectively, and decreased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups, respectively (p < 0.001). RNZ, AFL and ZFL groups were compared with each other in terms of cell viability levels, each group was significantly different from the other two (p < 0.001). Among these three groups, the cell viability level was the highest in the ZFL group and the lowest in the RNZ group. Similar results were observed in the RNZ + RSV, AFL + RSV and ZFL + RSV groups and in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups. Apoptosis (Fig. 4 B) levels in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p < 0.001). Apoptosis levels in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups; increase significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups respectively (p < 0.001). VEGF-A (Fig. 4 C) levels in the RNZ + RSV, AFL + RSV and ZFL + RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; decreased significantly compared to RSV group (p < 0.001). VEGF-A levels in the RNZ + RSV + Cob, AFL + RSV + Cob and ZFL + RSV + Cob groups were decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ + RSV, AFL + RSV and ZFL + RSV groups respectively (p < 0.001). VEGF-A levels in the RES group were decreased significantly compared to the Control group (p < 0.01). The RSV, RNZ, AFL and ZFL groups were compared among themselves, there was no statistically significant difference between the groups. Discussion Our study showed that resveratrol has antioxidant effects, reducing apoptosis and enhancing cell viability both alone and in combination with anti-VEGFs in vitro. According to our findings, resveratrol can support anti-VEGFs efficiency and reduce VEGF levels and can play a very effective role in combination therapy. In both atrophic and neovascular types of disease, progressive macular atrophy is closely related to vision loss [ 6 ]. The role of anti-VEGF agents in the progression of macular atrophy in patients receiving long-term anti-VEGF therapy is still the subject of many studies. There are studies indicating that anti-VEGF agents do not have adverse effects on the morphology, viability, density or proliferation of retinal cells; therefore, they do not increase the incidence or progression of macular atrophy and have a wide safety profile [ 26 , 36 – 38 ]. However, CATT and IVAN studies reported that long-term anti-VEGF therapy may play a role in the progression of macular atrophy [ 39 , 40 ]. Several studies have shown that the number of injections is a risk factor for the incidence and progression of macular atrophy [ 41 , 42 ]. Kuehlewein et. al. reported that long-term monthly intravitreal ranibizumab treatment is a risk factor for RPE atrophy [ 43 ]. Preclinical studies have shown that anti-VEGF drugs decrease proliferation and phagocytic capacity of cells in RPE cell culture and may cause an increase in apoptosis and a decrease in cell survival in newborn rabbit retinas [ 44 – 47 ]. Oxidative stress and damage play important roles in the pathogenesis of AMD [ 7 ]. It has been reported that the RPE gradually undergoes apoptosis under oxidative stress [ 48 ]. An investigation of the protective effects of resveratrol on RPE cells under oxidative stress conditions revealed that resveratrol can reduce apoptosis by inhibiting the expression of apoptotic genes Bcl-2 and caspase-3, increase cell viability and protect the cell against oxidative damage by modulating the activity of antioxidant enzymes in D407 cells [ 49 ]. Anti-VEGFs are the major therapy for AMD, but their role in macular atrophy progression has not been proven [ 39 , 40 , 50 ]. Although the possible atrophic effect of anti-VEGFs has not been well documented, we believe that this effect should not be ignored, and that some preventive approaches should be developed due to the irreversible nature of atrophy. In this context, applying a combined treatment involving the addition of molecules that can reduce macular atrophic progression in treatment regimens containing anti-VEGFs should be considered a viable alternative. Based on this idea, our observations aroused the thought that resveratrol, which has been shown in the literature to have antiapoptotic, antioxidant, anti-inflammatory and antiangiogenic properties, can serve this purpose. That’s why in our study resveratrol has been chosen as a combination molecule with anti-VEGFs in AMD treatment. Subramani et. al. reported an in-vitro study in which bevacizumab was combined with resveratrol [ 35 ]. In this study, various doses of bevacizumab and resveratrol combinations were applied to ARPE-19 cells and epithelial-mesenchymal transition rate was lower in cultures treated with these two agents compared to cultures treated with bevacizumab alone; moreover, phagocytosis was found to be increased in combined cultures compared to bevacizumab alone. Ranibizumab, aflibercept, ziv-aflibercept and resveratrol are the subject of various in-vivo and in-vitro studies, while no studies have evaluated the combined effects of these three molecules with resveratrol up to date. Our distinctive findings in the study present the first evidence to evaluate the combined effects of ranibizumab, aflibercept and ziv-aflibercept with resveratrol in-vitro. Intravitreal use of ziv-aflibercept is increasing daily due to its cost-effectiveness [ 5 ]. Many studies have reported positive results regarding the efficacy and safety of ziv-aflibercept [ 51 – 56 ]. Similarly, in our study, the viability of cells treated with ziv-aflibercept was greater than that of cells treated with ranibizumab or aflibercept according to MTT-plate reader analysis. On the other hand, there were no significant difference between ziv-aflibercept group and the ranibizumab and aflibercept groups, in accordance with the findings of our other analyses. These findings support previous studies that have reached favorable results on the efficacy and safety of ziv-aflibercept. Our study has several limitations. In this study, there was only one type of cell. Our study is an in-vitro study and does not evaluate in-vivo conditions such as the blood-retina barrier, immunological, circulatory, and inflammatory systems process and amount of agent reaching the tissue. ARPE-19 cells were exposed to the agents for only 48 hours. There was no repeated dosing and the dosing regimen was fixed in the study. In conclusion, macular atrophy is closely linked to visual loss in AMD patiens. Resveratrol can support cell survival by reducing oxidative stress and apoptosis on the combine treatment. Thus, resveratrol can decrease macular atrophy progression during long-term anti-VEGF therapy. Declarations Funding The study was supported by the Scientific Research Projects Coordination Unit of Süleyman Demirel University (SDU-BAP, Project No: TTU-2020-8124). Conflict of Interest The authors declare no conflict of interest. Author Contribution Conceptualization, M.A., O.K. and Ö.Ç.; methodology, O.K. and Ö.Ç.; software, O.K. and Ö.Ç.; validation, M.A., O.K., Ö.Ç., Ö.T., L.T and M.N.; formal analysis, O.K. and Ö.Ç.; investigation, M.A., O.K. and Ö.Ç.; resources, M.A. and O.K.; data curation, M.A., O.K. and Ö.Ç.; writing-original draft preparation, M.A., O.K., Ö.Ç., Ö.T., L.T and M.N.; writing—review and editing, M.A., O.K., Ö.Ç., Ö.T., L.T and M.N.; visualization, M.A., O.K. and Ö.Ç.; supervision, M.A., Ö.Ç., Ö.T. and L.T.; project administration, M.A., O.K. and Ö.Ç.; funding acquisition, M.A. All authors have read and agreed to the published version of the manuscript. Data Availability The datasets generated and analysed during the current study are not publicly available but are available from the corresponding author on reasonable request. References Flores R, Carneiro A, Vieira M, et al. 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Uǧuz AC, NazIroǧlu M, Espino J, et al. Selenium modulates oxidative stress-induced cell apoptosis in human myeloid HL-60 cells through regulation of calcium release and caspase-3 and – 9 activities. J Membr Biol. 2009;232:15–23. https://doi.org/10.1007/S00232-009-9212-2 . Öz A, Çelik Ö. Curcumin inhibits oxidative stress-induced TRPM2 channel activation, calcium ion entry and apoptosis values in SH-SY5Y neuroblastoma cells: Involvement of transfection procedure. Mol Membr Biol. 2016;33:76–88. https://doi.org/10.1080/09687688.2017.1318224 . Uğuz AC, Nazıroğlu M. Effects of selenium on calcium signaling and apoptosis in rat dorsal root ganglion neurons induced by oxidative stress. Neurochem Res. 2012;37:1631–8. https://doi.org/10.1007/S11064-012-0758-5 . Subramani M, Ponnalagu M, Krishna L, et al. Resveratrol reverses the adverse effects of bevacizumab on cultured ARPE-19 cells. Sci Rep. 2017;7. https://doi.org/10.1038/S41598-017-12496-Z . Abdelfattah NS, Al-Sheikh M, Pitetta S, et al. Macular Atrophy in Neovascular Age-Related Macular Degeneration with Monthly versus Treat-and-Extend Ranibizumab: Findings from the TREX-AMD Trial. Ophthalmology. 2017;124:215–23. https://doi.org/10.1016/J.OPHTHA.2016.10.002 . Schnichels S, Hagemann U, Januschowski K, et al. Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells. Br J Ophthalmol. 2013;97:917–23. https://doi.org/10.1136/BJOPHTHALMOL-2013-303130 . Saenz-De-Viteri M, Fernández-Robredo P, Hernández M, et al. Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions. Biochem Pharmacol. 2016;103:129–39. https://doi.org/10.1016/J.BCP.2015.12.017 . Grunwald JE, Daniel E, Huang J, et al. Risk of geographic atrophy in the comparison of age-related macular degeneration treatments trials. Ophthalmology. 2014;121:150–61. https://doi.org/10.1016/J.OPHTHA.2013.08.015 . Chakravarthy U, Harding SP, Rogers CA, et al. Alternative treatments to inhibit VEGF in age-related choroidal neovascularisation: 2-year findings of the IVAN randomised controlled trial. Lancet (London England). 2013;382:1258–67. https://doi.org/10.1016/S0140-6736(13)61501-9 . Daien V, Nguyen V, Essex RW, et al. Prevalence and characteristics of macular atrophy in eyes with neovascular age-related macular degeneration. A study from a long-term observational dataset: the Fight Retinal Blindness! project. Br J Ophthalmol. 2020;104:1064–9. https://doi.org/10.1136/BJOPHTHALMOL-2019-315055 . Lois N, McBain V, Abdelkader E, et al. Retinal pigment epithelial atrophy in patients with exudative age-related macular degeneration undergoing anti-vascular endothelial growth factor therapy. Retina. 2013;33:13–22. https://doi.org/10.1097/IAE.0B013E3182657FFF . Kuehlewein L, Dustin L, Sagong M, et al. Predictors of Macular Atrophy Detected by Fundus Autofluorescence in Patients With Neovascular Age-Related Macular Degeneration After Long-Term Ranibizumab Treatment. Ophthalmic Surg Lasers Imaging Retina. 2016;47:224–31. https://doi.org/10.3928/23258160-20160229-04 . Klettner A, Möhle F, Roider J. Intracellular bevacizumab reduces phagocytotic uptake in RPE cells. Graefes Arch Clin Exp Ophthalmol. 2010;248:819–24. https://doi.org/10.1007/S00417-010-1317-X . Klettner A, Tahmaz N, Dithmer M, et al. Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis. Br J Ophthalmol. 2014;98:1448–52. https://doi.org/10.1136/BJOPHTHALMOL-2014-305105 . Şahiner M, Bahar D, Öner A, et al. The Effects of Anti-Vascular Endothelial Growth Factor Drugs on Retinal Pigment Epithelial Cell Culture. Turkish J Ophthalmol. 2018;48:190–5. https://doi.org/10.4274/TJO.20270 . Cam D, Berk AT, Micili SC, et al. Histological and Immunohistochemical Retinal Changes Following the Intravitreal Injection of Aflibercept, Bevacizumab and Ranibizumab in Newborn Rabbits. Curr Eye Res. 2017;42:315–22. https://doi.org/10.3109/02713683.2016.1164190 . Chiang YW, Su CH, Sun HY, et al. Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells. Environ Toxicol. 2022;37:131–41. https://doi.org/10.1002/TOX.23384 . Yang Y, Wu ZZ, Cheng YL, et al. Resveratrol protects against oxidative damage of retinal pigment epithelium cells by modulating SOD/MDA activity and activating Bcl-2 expression. Eur Rev Med Pharmacol Sci. 2019;23:378–88. https://doi.org/10.26355/EURREV_201901_16786 . Semeraro F, Morescalchi F, Duse S, et al. Pharmacokinetic and Pharmacodynamic Properties of Anti-VEGF Drugs After Intravitreal Injection. Curr Drug Metab. 2015;16:572–84. https://doi.org/10.2174/1389200216666151001120831 . Dias JRDO, Badaró E, Novais EA, et al. Preclinical investigations of intravitreal ziv-aflibercept. Ophthalmic Surg Lasers Imaging Retina. 2014;45:577–84. https://doi.org/10.3928/23258160-20141118-15 . Chhablani J, Narayanan R, Mathai A, et al. SHORT-TERM SAFETY PROFILE OF INTRAVITREAL ZIV-AFLIBERCEPT. Retina. 2016;36:1126–31. https://doi.org/10.1097/IAE.0000000000000913 . Barmas-Alamdari D, D’Souza HS, Kapoor KG, Wagner AL. Intravitreal Ziv-Aflibercept: A Comprehensive Review. Semin Ophthalmol. 2019;34:420–35. https://doi.org/10.1080/08820538.2019.1641526 . HodjatJalali K, Mehravaran S, Faghihi H, et al. Intravitreal injection of ziv-aflibercept in the treatment of choroidal and retinal vascular diseases. J Curr Ophthalmol. 2017;29:228–31. https://doi.org/10.1016/J.JOCO.2017.01.005 . Mansour AM, Ashraf M, Dedhia CJ, et al. Long-term safety and efficacy of ziv-aflibercept in retinal diseases. Br J Ophthalmol. 2017;101:1374–6. https://doi.org/10.1136/BJOPHTHALMOL-2016-309724 . De Lima Farah J, Sano R, Maugéri IML, et al. Evaluation of aflibercept and ziv-aflibercept binding affinity to vascular endothelial growth factor, stability and sterility after compounding. Int J Retin Vitr. 2018;4. https://doi.org/10.1186/S40942-018-0143-X . Statements & Declarations Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4790772","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342681297,"identity":"4e3b91ee-8e4f-4c2b-98dd-9ea376a896a9","order_by":0,"name":"Onur KONUKCU","email":"","orcid":"","institution":"Süleyman Demirel University Research and Education Hospital","correspondingAuthor":false,"prefix":"","firstName":"Onur","middleName":"","lastName":"KONUKCU","suffix":""},{"id":342681301,"identity":"6e3ecce0-f65f-4c82-812f-2b27906e0fbc","order_by":1,"name":"Mehmet ARGUN","email":"","orcid":"","institution":"Süleyman Demirel University Research and Education Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"ARGUN","suffix":""},{"id":342681303,"identity":"a7db6b4c-a13b-4114-b460-f3cdb642111b","order_by":2,"name":"Ömer ÇELİK","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYPACCx4J9gYGCSCLsYFILRI8EjwHYFqYidPCICGRQKQWfrHDxz78qJCQkZz5/OFtHgYb2Q0H+I99wKdFcnZa8syeMxI80tI5xtY8DGnGGw4wM8/Ap8Xgdo4xM2ObBI+cdA6bNA/D4USQFrwOM7id/5mZ8R9Qi+TxZ0At/4nRksPMzNgAdJgEgxlQywHCWoB+MWbsOSbBI9mTY2w5xyDZeOZhZmO8Wvilkx8z/KixsZc4fvzhjTcVdrJ9xxsf49WC7k4gJi4mR8EoGAWjYBTgAwBW3D0KbaeBSAAAAABJRU5ErkJggg==","orcid":"","institution":"Süleyman Demirel University","correspondingAuthor":true,"prefix":"","firstName":"Ömer","middleName":"","lastName":"ÇELİK","suffix":""},{"id":342681305,"identity":"0e35689d-e989-4496-85e9-ef4775cb928b","order_by":3,"name":"Özlem TÖK","email":"","orcid":"","institution":"Süleyman Demirel University Research and Education Hospital","correspondingAuthor":false,"prefix":"","firstName":"Özlem","middleName":"","lastName":"TÖK","suffix":""},{"id":342681306,"identity":"27eb46c4-c135-4c02-b779-0ac95588870f","order_by":4,"name":"Levent TÖK","email":"","orcid":"","institution":"Süleyman Demirel University Research and Education Hospital","correspondingAuthor":false,"prefix":"","firstName":"Levent","middleName":"","lastName":"TÖK","suffix":""},{"id":342681308,"identity":"eafd167d-1870-4cf9-afad-895088a45bd0","order_by":5,"name":"Mustafa NAZIROĞLU","email":"","orcid":"","institution":"Süleyman Demirel University","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"NAZIROĞLU","suffix":""}],"badges":[],"createdAt":"2024-07-23 18:35:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4790772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4790772/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63011988,"identity":"b8a09c38-45c7-461b-b6fb-23318475f156","added_by":"auto","created_at":"2024-08-22 05:57:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1804881,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal microscope images (A) and findings (B, C, D, E, F). MitROS (B), MitDep (C) and CytROS (D) levels increased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; decreased significantly in the RSV group compared to the Control group (p\u0026lt;0.001). There was no significant difference in the RNZ, AFL and ZFL groups; in the RNZ+RSV, AFL+RSV and ZFL+RSV groups and in the RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups. Total number of cells (E) decreased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; increased significantly in the RSV group compared to the Control group (p\u0026lt;0.001). There was no significant difference in total number of cells in the RNZ, AFL and ZFL groups; in the RNZ+RSV, AFL+RSV and ZFL+RSV groups and in the RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups. Rate of cell death (F) increased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; decreased significantly in the RSV group compared to the Control group (p\u0026lt;0.001). There was no significant difference in the rate of cell death in the RNZ, AFL and ZFL groups; in the RNZ+RSV, AFL+RSV and ZFL+RSV groups and in the RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups.(\u003csup\u003ea\u003c/sup\u003ep\u0026lt; 0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt; 0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt; 0.001 vs. Cob group, \u003csup\u003eg\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ+RSV group, \u003csup\u003eh\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL+RSV group, \u003csup\u003ei\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL+RSV group). Each experiment was repeated at least six times.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4790772/v1/b4bfdef4d170422671cdd57d.png"},{"id":63011987,"identity":"8a483a36-af82-4acd-b25a-3d971ed9e7b1","added_by":"auto","created_at":"2024-08-22 05:57:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":391787,"visible":true,"origin":"","legend":"\u003cp\u003eMitDep (A) and CytROS (B) levels by plate reader. MitDep and CytROS level increased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; decreased significantly in the RSV group compared to the Control group (p\u0026lt;0.001). There was no significant difference in the MitDep and CytROS in the RNZ, AFL and ZFL groups; in the RNZ+RSV, AFL+RSV and ZFL+RSV groups and in the RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups. (\u003csup\u003ea\u003c/sup\u003ep\u0026lt; 0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt; 0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt; 0.001 vs. Cob group, \u003csup\u003eg\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ+RSV group, \u003csup\u003eh\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL+RSV group, \u003csup\u003ei\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL+RSV group). Each experiment was repeated at least six times.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4790772/v1/156b9ae74aad221c9b399170.png"},{"id":63011991,"identity":"4db7d02a-8f51-4f11-aa92-9c05a8fe108e","added_by":"auto","created_at":"2024-08-22 05:57:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639242,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase-3 (A), Caspase-8 (B) and Caspase-9 (C) activity levels by plate reader. Caspase-3 and Caspase-9 activity increased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; decreased significantly in the RSV group compared to the Control group (p\u0026lt;0.001). There was no statistical difference between RNZ, AFL and ZFL groups in terms of Caspase-3 and Caspase-9 activity. (for Fig.3A and Fig.3C; \u003csup\u003ea\u003c/sup\u003ep\u0026lt; 0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt; 0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt; 0.001 vs. Cob group) (for Fig.3B; \u003csup\u003ea\u003c/sup\u003ep\u0026lt; 0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt; 0.001 vs. Control and RSV groups, \u003csup\u003ec\u003c/sup\u003ep\u0026lt; 0.001 vs. Cob group). Each experiment was repeated at least six times.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4790772/v1/a66a5b99a0b15159c977c99a.png"},{"id":63012635,"identity":"d35a96b3-ba75-4510-b718-90bcefc209b3","added_by":"auto","created_at":"2024-08-22 06:05:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":572094,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability (A) levels, apoptosis (B) and VEGF-A (C) levels by plate reader. Cell viability level decreased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; increased significantly in the RSV group compared to the control group (p\u0026lt;0.001). Apoptosis level increased significantly in Cob, RNZ, AFL and ZFL groups compared to the Control group; decreased significantly in the RSV group compared to the control group (p\u0026lt;0.001). No statistically significant difference was observed between the RNZ, AFL and ZFL groups in terms of apoptosis levels. When the RNZ+RSV, AFL+RSV and ZFL+RSV groups were compared among themselves, no significant difference was found in terms of apoptosis levels. When the RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups were compared among themselves, no significant difference was observed in terms of apoptosis levels. VEGF-A level was found to be significantly increased in the Cob group compared to the Control group (p\u0026lt;0.001). VEGF-A levels decreased significantly in RSV, RNZ, AFL and ZFL groups compared to the Control group. There was no significant difference in VEGF-A levels between RNZ+RSV, AFL+RSV and ZFL+RSV groups. There was no significant difference in VEGF-A activity between RNZ+RSV+Cob, AFL+RSV+Cob and ZFL+RSV+Cob groups. (for Fig.4A; \u003csup\u003ea\u003c/sup\u003ep\u0026lt; 0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt; 0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt; 0.001 vs. Cob group, \u003csup\u003eg\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ+RSV group, \u003csup\u003eh\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL+RSV group, \u003csup\u003ei\u003c/sup\u003ep\u0026lt; 0.001 vs. ZFL+RSV group, \u003csup\u003ej\u003c/sup\u003ep\u0026lt; 0.001 vs. RNZ+RSV+Cob group, \u003csup\u003ek\u003c/sup\u003ep\u0026lt; 0.001 vs. AFL+RSV+Cob group) (for Fig4B; \u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.001 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt;0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt;0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt;0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt;0.001 compared to Cob group, \u003csup\u003eg\u003c/sup\u003ep\u0026lt;0.001 vs. RNZ+RSV group, \u003csup\u003eh\u003c/sup\u003ep\u0026lt;0.001 compared to AFL+RSV group, \u003csup\u003ei\u003c/sup\u003ep\u0026lt;0.001 compared to ZFL+RSV group) (for Fig4C; \u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.001 vs. Control group, *p\u0026lt;0.01 vs. Control group, \u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.001 vs. RSV group, \u003csup\u003ec\u003c/sup\u003ep\u0026lt;0.001 vs. RNZ group, \u003csup\u003ed\u003c/sup\u003ep\u0026lt;0.001 vs. AFL group, \u003csup\u003ee\u003c/sup\u003ep\u0026lt;0.001 vs. ZFL group, \u003csup\u003ef\u003c/sup\u003ep\u0026lt;0.001 compared to Cob group, \u003csup\u003eg\u003c/sup\u003ep\u0026lt;0.001 vs. RNZ+RSV group, \u003csup\u003eh\u003c/sup\u003ep\u0026lt;0.001 compared to AFL+RSV group, \u003csup\u003ei\u003c/sup\u003ep\u0026lt;0.001 compared to ZFL+RSV group). Each experiment was repeated at least six times.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4790772/v1/3d4d427b8e75b20d11fd2e1d.png"},{"id":86578663,"identity":"b062968b-2180-45f2-8698-4ee0ca470f21","added_by":"auto","created_at":"2025-07-12 19:01:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4710700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4790772/v1/c7a288fa-6b75-46d2-bdeb-c7495588e911.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An In-vitro Evaluation of a New Approach in AMD: Effects of the Combination of Resveratrol and Anti- VEGFs on ARPE-19 Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAge-related macular degeneration (AMD) is a chronic condition that leads to loss of central vision in the elderly individuals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. AMD is the most common cause of blindness in developed countries for people older than 60 years, accounting for 8.7% of all blindness cases globally and is projected to impact over 280\u0026nbsp;million people by 2040 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Anti-VEGF agents are the most successful treatment for AMD, as a VEGF-A is essential for the formation and growth of choroidal neovascularization, which is a major cause of vision loss in neovascular AMD [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The agents used for this purpose are ranibizumab, aflibercept and bevacizumab [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Ziv-aflibercept, which is not approved for eye use, has been used more often because of its cost-effectiveness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMacular atrophy is associated with vision loss in patients with AMD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The macular atrophy progresses even when patients receive anti-VEGF therapy. The cause of macular atrophy progression is unknown. Oxidative stress and apoptosis may contribute to macular cell death, which leads to macular atrophy progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The combination of anti-VEGFs with some molecules that reduce macular atrophy progression may improve the treatment options for AMD.\u003c/p\u003e \u003cp\u003eResveratrol (3,5,4\u0026prime;-trihydroxystilbene) is a polyphenolic phytoalexin produced from phenyl alanine by stilbene synthase in plants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It attracted medical interest with the \u0026ldquo;French Paradox\u0026rdquo;, which proposes that red wine with high levels of resveratrol is good for cardiovascular health [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Resveratrol affects various physiological processes, such as oxidative stress, inflammation, the cell cycle and proliferation, metastasis, apoptosis, and angiogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has a strong antioxidant and anti-inflammatory effect [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Resveratrol has been studied for its cardioprotective, neuroprotective, chemotherapeutic, and anti-aging effect [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Resveratrol has shown antiangiogenic, antiplatelet and antiproliferative effects [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In-vitro studies have demonstrated that resveratrol can protect retinal pigment epithelium cells (RPE) from oxidative stress and apoptosis caused by sodium iodate and autoimmunity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Resveratrol has been reported to protect against light-induced retinal degeneration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been shown that it inhibits VEGF secretion induced by hypoxia and inflammatory cytokines such as IFN-γ, TNF-α, IL-1β, and TGF-β [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, resveratrol may be a potential therapeutic agent for AMD, in which inflammation, cell death-related atrophy and VEGF-based neovascularization play key roles in its pathogenesis.\u003c/p\u003e \u003cp\u003eThe aim of this study was to examine the effects of adding resveratrol to anti-VEGFs (ranibizumab, aflibercept, ziv-aflibercept) on ARPE-19 cells in-vitro. We measured and analysed intracellular and mitochondrial ROS levels, mitochondrial membrane depolarization, caspase-3, caspase-8 and caspase-9 activities, cell viability, apoptosis, and VEGF-A levels in the anti-VEGF treatment groups with or without resveratrol to determine the effects of resveratrol in combination treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Solutions\u003c/h2\u003e \u003cp\u003eThe human retinal pigment epithelium cell line (ARPE-19) was acquired from American Type Cell Culture (ATCC-Virginia, USA, ATCC Number: CRL-2302). Cell culture medium was obtained from Sigma Aldrich (Missouri, USA). APOPercentageTM assay kit was obtained from Biocolor (Belfast, Northern Ireland). Human Vascular Endothelial Cell Growth Factor A (VEGF-A) ELISA kit was procured from Bioassay Technology Lab (Shanghai, China, Cat. No. E0050Hu). All reagents were of analytical grade. Each experiment was performed at least six times.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eARPE-19 cells were grown in a concoction medium consisting of 1:1 ratio of Dulbecco\u0026rsquo;s Modified Eagle Medium and Ham\u0026rsquo;s F12 medium complemented with 10% fetal bovine serum (Biochrom, Germany) and 1% penicillin-streptomycin mixture (Biochrom, Germany) in accordance with the manufacturer's instructions. Cells were used in passages between 2\u0026ndash;12. The cell culture operations were implemented at 37\u0026deg;C in a humidified incubator (Heal Force HF90, Japan) with 5% CO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe Experimental Design\u003c/h2\u003e \u003cp\u003eThe final volume of growth medium per flask in all groups was determined as 4 ml which is the physiological volume of vitreous and the incubation dose of anti-VEGF drugs was ascertained according to Malik et al.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The study groups and treatments were followed as:\u003c/p\u003e \u003cp\u003eControl: ARPE-19 cells were incubated for 48 h in a standard growth medium and under the conditions described above.\u003c/p\u003e \u003cp\u003eCobalt Chloride (CoCl\u003csub\u003e2\u003c/sub\u003e) Group (Cob): The cells were incubated for 48 h. In the 24th h of incubation, the cells were treated with CoCl\u003csub\u003e2\u003c/sub\u003e (200 \u0026micro;M) without refreshing the growth medium.\u003c/p\u003e \u003cp\u003eResveratrol Group (RSV): The cells were incubated with RSV (100 \u0026micro;M) for 48 h.\u003c/p\u003e \u003cp\u003eRanibizumab Group (RNZ): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) for 48 h.\u003c/p\u003e \u003cp\u003eAflibercept Group (AFL): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) for 48 h.\u003c/p\u003e \u003cp\u003eZiv-aflibercept Group (ZFL): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) for 48 h.\u003c/p\u003e \u003cp\u003eRanibizumab\u0026thinsp;+\u0026thinsp;Resveratrol Group (RNZ\u0026thinsp;+\u0026thinsp;RSV): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h.\u003c/p\u003e \u003cp\u003eAflibercept\u0026thinsp;+\u0026thinsp;Resveratrol Group (AFL\u0026thinsp;+\u0026thinsp;RSV): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h.\u003c/p\u003e \u003cp\u003eZiv-aflibercept\u0026thinsp;+\u0026thinsp;Resveratrol Group (ZFL\u0026thinsp;+\u0026thinsp;RSV): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h.\u003c/p\u003e \u003cp\u003eRanibizumab\u0026thinsp;+\u0026thinsp;Resveratrol\u0026thinsp;+\u0026thinsp;Cobalt Chloride Group (RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob): The cells were incubated with a clinical dose of RNZ (0.5 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h. In the 24th h of incubation, the cells were treated with CoCl\u003csub\u003e2\u003c/sub\u003e (200 \u0026micro;M) without refreshing the growth medium.\u003c/p\u003e \u003cp\u003eAflibercept\u0026thinsp;+\u0026thinsp;Resveratrol\u0026thinsp;+\u0026thinsp;Cobalt Chloride Group (AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob): The cells were incubated with a clinical dose of AFL (2 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h. In the 24th h of incubation, the cells were treated with CoCl\u003csub\u003e2\u003c/sub\u003e (200 \u0026micro;M) without refreshing the growth medium.\u003c/p\u003e \u003cp\u003eZiv-aflibercept\u0026thinsp;+\u0026thinsp;Resveratrol\u0026thinsp;+\u0026thinsp;Cobalt Chloride Group (ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob): The cells were incubated with a clinical dose of ZFL (2 mg/4 ml) and RSV (100 \u0026micro;M) for 48 h. In the 24th h of incubation, the cells were treated with CoCl\u003csub\u003e2\u003c/sub\u003e (200 \u0026micro;M) without refreshing the growth medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConfocal Microscopy Analysis\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial ROS Generation (MitROS), Mitochondrial Membrane Depolarization (MitDep) and Cytosolic ROS Generation (CytROS)\u003c/h2\u003e \u003cp\u003eChanges in mitochondrial ROS generation (MitROS), mitochondrial membrane depolarization (MitDep) and cytosolic ROS generation (CytROS) in ARPE-19 cells were observed in an LSCM/800 instrument with a Plan-Apochromat 20\u0026times;/0.8 objective (Zeiss, Germany). To assess MitROS and CytROS levels; MitoTracker Red CM-H2Xros (MtTrk, 1 \u0026micro;M for 30 min) and DCFH/DA (1 \u0026micro;M for 30 min) probes were applied respectively. The cells were exposed to the fluorescent probe JC-1 (2 \u0026micro;M) for 30 min to determining of MitDep. MtTrk (561 nm), JC-1 (488 nm) and DCFH/DA (488 nm) were activated with a diode argon laser. The ZEN program was used to measure fluorescence scattering as arbitrary unit (a.u.) in the captured red (MtTrk), orange (JC-1) and green (DCFH/DA) images [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTotal Number of Cells and Rate of Cell Death\u003c/h2\u003e \u003cp\u003eThe cell death ratio was assessed in the images obtained after staining with Hoechst 33342 (Thermo Fisher Sci.) and propidium iodide (PI; Thermo Fisher Sci.). Hoechst 33342 (8.1 \u0026micro;M) and PI (1.5 \u0026micro;M) dyes were applied to the dishes with bottom glass. The cells were incubated for 20 min with PI and Hoechst 33342, and then the red and blue images were captured by the LSCM/800. The ratio of PI-positive (dead) cells was calculated by individually counting the number of blue and red cells using the ZEN program [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlate Analysis\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial Membrane Depolarization (MitDep) and Cytosolic ROS Generation (CytROS)\u003c/h2\u003e \u003cp\u003eThe oscillations in MitDep and CytROS were measured by an automatic plate reader (Infinite 200 PRO, Tecan Life Sci. M\u0026auml;nnedorf, Switzerland) with JC-1 and DHR-123 dyes, respectively. The fluorescence scattering density variations of JC-1 and DHR-123 were expressed as a percentage of the control after computing the fluorescence units/mg protein [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3, 8 and 9\u003c/h2\u003e \u003cp\u003eThe activities of Caspase-3, Caspase-8 and Caspase-9 were detected by using the passive fluorogenic substrates Ac-DEVD-AMC, Ac-IETD-AFC, and Ac-LEHD-AFC (Bachem AG., Bubendorf, Switzerland) respectively. The levels of AMC and AFC which are specifically cleaved by active Caspase-3, Caspase-8 and Caspase-9 were measured by Infinite 200 PRO (Ext: 360\u0026ndash;400 nm, Ems: 460\u0026ndash;505 nm). The results are expressed as a percentage of the control [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability and Apoptosis\u003c/h2\u003e \u003cp\u003eThe MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test is a method used to measure cell viability. The principle of the test is that only living cells can reduce a yellow, water-soluble tetrazolium salt to a purple, water-insoluble formazan crystals. Dead cells cannot perform this enzymatic reaction. After drug treatment, the cells were separated and MTT dye was added to the tubes. The cells were incubated in a humidified incubator for 90 min at 37\u0026deg;C. The supernatant was discarded and 150 \u0026micro;L of DMSO was used to solubilize the formazan crystals. An automated multiplate reader (Tecan Infinite 200 Pro) was used to measured the optical densities at 490 and 650 nm (as reference wavelength) and the results were expressed as the fold change relative to control as experiment/control [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The APOPercentage Apoptosis Kit (Biocolor Ltd., Northern Ireland) was used to assess the level of apoptosis according to a protocol described elsewhere. The results were shown as a percentage of the control [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVEGF-A Levels\u003c/h2\u003e \u003cp\u003eVEGF-A levels were determined according to the manufacturer\u0026rsquo;s instructions by using a commercial ELISA (Cat.No: E0050Hu, BT Lab, China). The curved line had an R\u003csup\u003e2\u003c/sup\u003e of 0.998. The results were given as pg/ml [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConfocal Microscopy Analysis Results\u003c/h2\u003e \u003cp\u003eThe confocal microscopy images and findings are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe MitROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), MitDep (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and CytROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups respectively; increased significantly compared to the RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). MitROS, MitDep and CytROS levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe total number of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups increased significantly compared to RNZ, AFL and ZFL groups respectively; decreased significantly compared to RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The total number of cells in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups increased significantly compared to the Cob, RNZ, AFL and ZFL groups and decreased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe rate of cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The rates of cell death in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePlate Reader Analysis Results\u003c/h2\u003e \u003cp\u003eThe MitDep and CytROS levels measured by a plate reader are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eMitDep (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and CytROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared with the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). MitDep and CytROS levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eCaspase-3, Caspase-8 and Caspase-9 activity levels are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eCaspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and Caspase-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) activity in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared with the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Caspase-3 and Caspase-9 activity in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups, there was no statistically significant difference.\u003c/p\u003e \u003cp\u003eCaspase-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) activity increased significantly in the Cob, RNZ, AFL, ZFL, RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV, ZFL\u0026thinsp;+\u0026thinsp;RSV, RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups compared to the Control and RSV groups; decreased significantly in the RSV group compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was no significant difference in Caspase-8 activity between the RNZ, AFL, ZFL, RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV, ZFL\u0026thinsp;+\u0026thinsp;RSV, RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups.\u003c/p\u003e \u003cp\u003eCell viability, apoptosis and VEGF-A levels are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eCell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) level in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups increased significantly compared to the RNZ, AFL and ZFL groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001); decreased significantly compared to RSV group. It was determined that cell viability was higher in the groups in which anti-VEGF agents and resveratrol were combined compared to the groups in which anti-VEGF agents were used alone. The cell viability level in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups increased significantly compared to the Cob group; increased significantly compared to the RNZ, AFL and ZFL groups, respectively, and decreased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). RNZ, AFL and ZFL groups were compared with each other in terms of cell viability levels, each group was significantly different from the other two (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Among these three groups, the cell viability level was the highest in the ZFL group and the lowest in the RNZ group. Similar results were observed in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups and in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups.\u003c/p\u003e \u003cp\u003eApoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; increased significantly compared to the RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Apoptosis levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups decreased significantly compared to the Cob, RNZ, AFL and ZFL groups; increase significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eVEGF-A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups decreased significantly compared to the RNZ, AFL and ZFL groups, respectively; decreased significantly compared to RSV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). VEGF-A levels in the RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob groups were decreased significantly compared to the Cob, RNZ, AFL and ZFL groups and increased significantly compared to the RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV and ZFL\u0026thinsp;+\u0026thinsp;RSV groups respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). VEGF-A levels in the RES group were decreased significantly compared to the Control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The RSV, RNZ, AFL and ZFL groups were compared among themselves, there was no statistically significant difference between the groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study showed that resveratrol has antioxidant effects, reducing apoptosis and enhancing cell viability both alone and in combination with anti-VEGFs in vitro. According to our findings, resveratrol can support anti-VEGFs efficiency and reduce VEGF levels and can play a very effective role in combination therapy.\u003c/p\u003e \u003cp\u003eIn both atrophic and neovascular types of disease, progressive macular atrophy is closely related to vision loss [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The role of anti-VEGF agents in the progression of macular atrophy in patients receiving long-term anti-VEGF therapy is still the subject of many studies. There are studies indicating that anti-VEGF agents do not have adverse effects on the morphology, viability, density or proliferation of retinal cells; therefore, they do not increase the incidence or progression of macular atrophy and have a wide safety profile [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, CATT and IVAN studies reported that long-term anti-VEGF therapy may play a role in the progression of macular atrophy [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Several studies have shown that the number of injections is a risk factor for the incidence and progression of macular atrophy [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Kuehlewein et. al. reported that long-term monthly intravitreal ranibizumab treatment is a risk factor for RPE atrophy [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Preclinical studies have shown that anti-VEGF drugs decrease proliferation and phagocytic capacity of cells in RPE cell culture and may cause an increase in apoptosis and a decrease in cell survival in newborn rabbit retinas [\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Oxidative stress and damage play important roles in the pathogenesis of AMD [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It has been reported that the RPE gradually undergoes apoptosis under oxidative stress [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. An investigation of the protective effects of resveratrol on RPE cells under oxidative stress conditions revealed that resveratrol can reduce apoptosis by inhibiting the expression of apoptotic genes Bcl-2 and caspase-3, increase cell viability and protect the cell against oxidative damage by modulating the activity of antioxidant enzymes in D407 cells [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnti-VEGFs are the major therapy for AMD, but their role in macular atrophy progression has not been proven [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Although the possible atrophic effect of anti-VEGFs has not been well documented, we believe that this effect should not be ignored, and that some preventive approaches should be developed due to the irreversible nature of atrophy. In this context, applying a combined treatment involving the addition of molecules that can reduce macular atrophic progression in treatment regimens containing anti-VEGFs should be considered a viable alternative. Based on this idea, our observations aroused the thought that resveratrol, which has been shown in the literature to have antiapoptotic, antioxidant, anti-inflammatory and antiangiogenic properties, can serve this purpose. That\u0026rsquo;s why in our study resveratrol has been chosen as a combination molecule with anti-VEGFs in AMD treatment. Subramani et. al. reported an in-vitro study in which bevacizumab was combined with resveratrol [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, various doses of bevacizumab and resveratrol combinations were applied to ARPE-19 cells and epithelial-mesenchymal transition rate was lower in cultures treated with these two agents compared to cultures treated with bevacizumab alone; moreover, phagocytosis was found to be increased in combined cultures compared to bevacizumab alone. Ranibizumab, aflibercept, ziv-aflibercept and resveratrol are the subject of various in-vivo and in-vitro studies, while no studies have evaluated the combined effects of these three molecules with resveratrol up to date. Our distinctive findings in the study present the first evidence to evaluate the combined effects of ranibizumab, aflibercept and ziv-aflibercept with resveratrol in-vitro.\u003c/p\u003e \u003cp\u003eIntravitreal use of ziv-aflibercept is increasing daily due to its cost-effectiveness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Many studies have reported positive results regarding the efficacy and safety of ziv-aflibercept [\u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Similarly, in our study, the viability of cells treated with ziv-aflibercept was greater than that of cells treated with ranibizumab or aflibercept according to MTT-plate reader analysis. On the other hand, there were no significant difference between ziv-aflibercept group and the ranibizumab and aflibercept groups, in accordance with the findings of our other analyses. These findings support previous studies that have reached favorable results on the efficacy and safety of ziv-aflibercept.\u003c/p\u003e \u003cp\u003eOur study has several limitations. In this study, there was only one type of cell. Our study is an in-vitro study and does not evaluate in-vivo conditions such as the blood-retina barrier, immunological, circulatory, and inflammatory systems process and amount of agent reaching the tissue. ARPE-19 cells were exposed to the agents for only 48 hours. There was no repeated dosing and the dosing regimen was fixed in the study.\u003c/p\u003e \u003cp\u003eIn conclusion, macular atrophy is closely linked to visual loss in AMD patiens. Resveratrol can support cell survival by reducing oxidative stress and apoptosis on the combine treatment. Thus, resveratrol can decrease macular atrophy progression during long-term anti-VEGF therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Scientific Research Projects Coordination Unit of S\u0026uuml;leyman Demirel University (SDU-BAP, Project No: TTU-2020-8124).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, M.A., O.K. and \u0026Ouml;.\u0026Ccedil;.; methodology, O.K. and \u0026Ouml;.\u0026Ccedil;.; software, O.K. and \u0026Ouml;.\u0026Ccedil;.; validation, M.A., O.K., \u0026Ouml;.\u0026Ccedil;., \u0026Ouml;.T., L.T and M.N.; formal analysis, O.K. and \u0026Ouml;.\u0026Ccedil;.; investigation, M.A., O.K. and \u0026Ouml;.\u0026Ccedil;.; resources, M.A. and O.K.; data curation, M.A., O.K. and \u0026Ouml;.\u0026Ccedil;.; writing-original draft preparation, M.A., O.K., \u0026Ouml;.\u0026Ccedil;., \u0026Ouml;.T., L.T and M.N.; writing\u0026mdash;review and editing, M.A., O.K., \u0026Ouml;.\u0026Ccedil;., \u0026Ouml;.T., L.T and M.N.; visualization, M.A., O.K. and \u0026Ouml;.\u0026Ccedil;.; supervision, M.A., \u0026Ouml;.\u0026Ccedil;., \u0026Ouml;.T. and L.T.; project administration, M.A., O.K. and \u0026Ouml;.\u0026Ccedil;.; funding acquisition, M.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFlores R, Carneiro A, Vieira M, et al. 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Int J Retin Vitr. 2018;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/S40942-018-0143-X\u003c/span\u003e\u003cspan address=\"10.1186/S40942-018-0143-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aflibercept, AMD, Ranibizumab, Resveratrol, Ziv-aflibercept","lastPublishedDoi":"10.21203/rs.3.rs-4790772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4790772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to determine how resveratrol combination separately with the anti-VEGF agents ranibizumab, aflibercept and ziv-aflibercept affects ARPE-19 cells in vitro.\u003c/p\u003e \u003cp\u003eThe cells were assigned to twelve groups as follows: Control, Cobalt Chloride (Cob), Resveratrol (RSV), Ranibizumab (RNZ), Aflibercept (AFL), Ziv-aflibercept (ZFL), RNZ\u0026thinsp;+\u0026thinsp;RSV, AFL\u0026thinsp;+\u0026thinsp;RSV, ZFL\u0026thinsp;+\u0026thinsp;RSV, RNZ\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob, AFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob and ZFL\u0026thinsp;+\u0026thinsp;RSV\u0026thinsp;+\u0026thinsp;Cob. The Control group was incubated for 48 hours with no treatment, while the remaining groups received RSV, RNZ, AFL or ZFL (alone or in combination) for 24 hours and then the cells in the relevant groups were exposed to CoCl\u003csub\u003e2\u003c/sub\u003e for 24 hours more. Mitochondrial reactive oxygen species (MitROS), cytosolic reactive oxygen species (CytROS), mitochondrial membrane depolarization (MitDep), caspase-3, caspase-8, caspase-9, cell viability, apoptosis and VEGF-A levels were assessed by confocal microscopy, plate reader and ELISA techniques.\u003c/p\u003e \u003cp\u003eResveratrol, alone or in combination with anti-VEGF\u0026rsquo;s, significantly decreased the levels of MitROS, MitDep, CytROS, caspase-3, caspase-8 and caspase-9 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Resveratrol also increased cell viability and decreased apoptosis and VEGF-A levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eAccording to our findings, combining resveratrol with anti-VEGFs may have a beneficial therapeutic effect on the treatment of AMD.\u003c/p\u003e","manuscriptTitle":"An In-vitro Evaluation of a New Approach in AMD: Effects of the Combination of Resveratrol and Anti- VEGFs on ARPE-19 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 05:57:07","doi":"10.21203/rs.3.rs-4790772/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"406099a9-d45c-4d95-a718-37a8a2655e71","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-12T18:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 05:57:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4790772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4790772","identity":"rs-4790772","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}