To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress.
We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 µM hydrogen peroxide (H2O2) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction.
VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 µM H2O2, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF.
When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these
The hypothesis that a specific substance acts on neovascularization in retinal diseases was first proposed in 1956 [
Differentiation of RGC-5 cells was used in this study. The RGC-5 cell line selectively retained the retinal ganglion cells in the mouse and was infected with the R-virus to maintain retinal ganglion cell characteristics [
The expression of VEGF, VEGFR-1, and VEGFR-2 was assessed by real-time polymerase chain reaction (PCR) and Western blot (
cDNA was synthesized from the extracted total RNA according to the manufacturer's protocol using the SuperScript III First-Strand Synthesis System for real-time PCR (Gibco, Grand Island, NY, USA). Briefly, cultured RGC-5 cells were floated with trypsin and total RNA was extracted using RNeasy mini kit (Qiagen, Valencia, CA, USA). A combination of 1 µL of extracted total RNA, 1 µL of 50 µM oligo (dT), 1 µL of 10 mM deoxynucleoside triphosphate (dNTP) mix and diethyl pyrocarbonate (DEPC)-treated water for a total volume of 10 µL was incubated at 65℃ for 5 minutes. The mixture was then put on ice for 1 minute to stop the reaction. Next, 2 µL of 10 × reverse transcription (RT) buffer, 4 µL of 25 mM MgCl2, 2 µL of 0.1 M dithiothreitol (DTT), 1 µL of RNaseOUT (40 U/µL) and 1 µL of SuperScript III RT (200 U/µL) was added for a total volume of 20 µL. Thereafter, the cells were incubated at 50℃ for 50 minutes and incubated at 85℃ for 5 minutes to stop the reaction, and then the tubes were placed on ice. The reaction mixture was collected by light centrifugation and 1 µL of RNase H was added and incubated at 37℃ for 20 minutes to remove residual RNA. The resulting cDNA was stored at −20℃ until further processing.
Real-time PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen). A mixture of 100 ng of cDNA, 2 µL of 20 µM primers, 25 µL of master mix, and DEPC-treated water was added to each sample to make a 50 µL mixture. The primer sequences are listed in
Each primer product was identified via melting curve analysis. The temperature conditions of the amplification process are as follows. Cycle 1, 95℃ for 3 minutes; cycle 2 (50 cycles; step 1, 95℃ for 10 seconds; step 2, 55℃ for 45 seconds); cycle 3, 95℃ for 1 minute; cycle 4, 55℃ for 1 minute; cycle 5 (80 cycles; step 1, 55℃ for 10 seconds; step 2, increase set point temperature after 2nd cycle by 0.5℃). Each mRNA level was calculated using the 2-ΔCt method based on the Ct value using β-actin as the reference housekeeping gene.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed with 10%–15% sodium dodecyl sulfate-polyacrylamide gel and 50 µg of cell lysate was loaded in each well. After electrophoresis, the cells were electro-transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA) under 200 mA for 2 hours. Three percent of BSA (AMRESCO, Solon, OH, USA) was used to inhibit nonspecific binding. After that, the primary antibody was added, reacted at room temperature for 1 hour, washed and secondary antibody was used. One of the ECL peroxidase-labeled anti-rabbit (Amersham, Piscataway, NJ, USA) or ECL peroxidase-labeled anti-mouse (Amersham) was selected for the primary antibody host, and then sensitized using Amersham ECL Western Blotting Detection Reagents (Amersham).
The differentiated RGC-5 cells were treated with H2O2 (800 µM) for 24 hours to induce oxidative stress. Cell viability was assessed using lactate dehydrogenase (LDH) assay. The amount of LDH was measured via colorimetric assay (Calbiochem-Novabiochem, San Diego, CA, USA), and whole cells were frozen. In comparison with the amount of LDH measured after death, the degree of destruction was evaluated. Serial dilutions of commercially available bevacizumab (Avastin; Genentech, San Francisco, CA, USA) were performed to obtain treatment concentrations of 0.01, 0.1, and 1 mg/mL.
Data are expressed as the mean ± standard deviation of at least three different experiments performed from separate cell preparations, and at least triplicate determinations were performed for each experiment. Statistical tests to determine the difference between groups were performed by Mann-Whitney
Oxidative stress was induced in RGC-5 cells treated for 24 hours using 800 µM H2O2. At that time, the RGC-5 cells were simultaneously treated with the anti-VEGF antibody, bevacizumab (0, 0.01, 0.1, and 1 mg), and the effect of this treatment on the survival was evaluated by LDH assay. We confirmed that the cell damage increased as the concentration of the bevacizumab increased. In the absence of oxidative stress, RGC-5 cytotoxicity increased following treatment with 1 mg bevacizumab, which was higher than oxidative stress-induced cytotoxicity (
The expression of VEGF, VEGFR-1, and VEGFR-2 in bevacizumab-treated RGC-5 cells was confirmed by Western blot analysis. In addition, the expression of VEGF, VEGFR-1, and VEGFR-2 increased as the concentration of bevacizumab increased, and the expression of VEGF, VEGFR-1, and VEGFR-2 increased in RGC-5 cells treated by H2O2 alone (
A number of recent studies have shown that bevacizumab injection therapy in the vitreous cavity has a remarkable therapeutic effect in many retinal vascular diseases including exudative age-related macular degeneration, diabetic retinopathy, and neovascular glaucoma [
Although intraocular pressure reduction still remains the mainstay of glaucoma therapy, recent studies have suggested that intraocular pressure reduction alone cannot prevent irreversible damage to retinal ganglion cells. Neuroprotection is a strategy for glaucoma treatment, and a number of studies have addressed the factors affecting the survival and death of the retinal ganglion cells [
In this study, we investigated the effects of bevacizumab concentration on RGC-5 cell survival under oxidative stress. We found that cytotoxicity increased when bevacizumab was applied to the differentiated RGC-5 cell line, and cell damage increased with increasing bevacizumab concentration. In addition, even in the absence of oxidative stress, when bevacizumab was applied to the RGC-5 cell line, the cytotoxicity of the cell line increased, and was greater than that from oxidative stress (
This study shows that a high concentration of bevacizumab increases cell death in retinal ganglion cells. However, since this experiment was not performed
This study is expected to provide a basis for the safe and effective use of bevacizumab in various ophthalmic diseases including glaucoma. In the future, bevacizumab should be used at the minimum concentration that does not cause cytotoxicity. In addition, intravitreal injection of bevacizumab for the treatment of retinal vascular disease often needs to be repeated rather than administered as a single treatment. Therefore, studies on the benefits and risks of long-term treatment according to the frequency of injection are needed to prevent potential adverse effects of indiscriminate bevacizumab treatment. In particular, considering the fact that bevacizumab treatment may have a negative effect on eyes with optic nerve damage such as glaucoma, treatment should be performed according to individual characteristics and more attention should be focused on these conditions. In addition, long-term large-scale clinical trials and animal models are needed in the future to evaluate the efficacy and safety of bevacizumab therapy.
This study was supported by a faculty research grant of Yonsei University College of Medicine for 2008 (research project No. 6-2008-0201).
No potential conflict of interest relevant to this article was reported.
PCR = polymerase chain reaction; VEGF = vascular endothelial growth factor; VEGFR = VEGF receptor.