Korean J Ophthalmol.  2017 Aug;31(4):360-365. 10.3341/kjo.2017.0054.

Effect of Anti-vascular Endothelial Growth Factor Antibody on the Survival of Cultured Retinal Ganglion Cells

• Lee JM
• Bae HW
• Lee SY
• Seong GJ
• Kim CY

• KMID: 2385586
• DOI: http://doi.org/10.3341/kjo.2017.0054

Abstract

PURPOSE
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.
METHODS
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 (Hâ‚‚Oâ‚‚) 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.
RESULTS
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 Hâ‚‚Oâ‚‚, 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.
CONCLUSIONS
When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.

Keyword

Anti-vascular endothelial growth factor; Bevacizumab; Oxidative stress; Retinal ganglion cell; RGC-5

MeSH Terms

Bevacizumab
Blotting, Western
Endothelial Growth Factors*
Hydrogen Peroxide
In Vitro Techniques
L-Lactate Dehydrogenase
Oxidative Stress
Real-Time Polymerase Chain Reaction
Receptors, Vascular Endothelial Growth Factor
Retinal Ganglion Cells*
Retinaldehyde*
Staurosporine
Survival Rate
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2
Bevacizumab
Endothelial Growth Factors
Hydrogen Peroxide
L-Lactate Dehydrogenase
Receptors, Vascular Endothelial Growth Factor
Retinaldehyde
Staurosporine
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2

Figure

• 

  Fig. 1 Expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1 and VEGFR-2 assessed by real-time polymerase chain reaction. VEGF, VEGFR-1, and VEGFR-2 are all expressed in differentiated retinal ganglion cell-5 cells.

• 

  Fig. 2 Effects of bevacizumab on the survival of retinal ganglion cell (RGC)-5 cells under oxidative stress with 800 µM H2O2 for 24 hours. As the concentration of bevacizumab increases, the RGC-5 death rate increases. With the presence of bevacizumab, cell death increased without oxidative stress. Values are presented as mean ± standard deviation (*values are significantly different from the control; p < 0.05 by Mann-Whitney U-test).

• 

  Fig. 3 Western blot analysis of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1 and VEGFR-2 in retinal ganglion cell (RGC)-5 cells treated with bevacizumab. The expression of VEGF, VEGFR-1, and VEGFR-2 increased with the increase in bevacizumab concentration, and the expression of VEGF, VEGFR-1, and VEGFR-2 was increased in RGC-5 cells treated with H2O2. 1, control; 2, bevacizumab 1 mg; 3, H2O2 800 µM; 4, bevacizumab 0.01 mg + H2O2 800 µM; 5, bevacizumab 0.1 mg + H2O2 800 µM; 6, bevacizumab 1 mg + H2O2 800 µM.

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