Korean J Ophthalmol.  2018 Aug;32(4):328-338. 10.3341/kjo.2017.0079.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells

• Chae JB
• Rho CR
• Shin JA
• Lyu J
• Kang S

• KMID: 2418154
• DOI: http://doi.org/10.3341/kjo.2017.0079

Abstract

PURPOSE
Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells.
METHODS
Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept.
RESULTS
Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells.
CONCLUSIONS
Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Keyword

Aflibercept; Aging; Bevacizumab; Ranibizumab; Retinal pigment epithelial cells

MeSH Terms

Aging
Bevacizumab*
Biomarkers
Endothelial Growth Factors
Epithelial Cells*
Humans
Phagocytosis
Ranibizumab*
Retinaldehyde*
Vascular Endothelial Growth Factor A
Bevacizumab
Biomarkers
Endothelial Growth Factors
Ranibizumab
Retinaldehyde
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 (A) Expression of retinal pigment epithelial (RPE) cell specific markers in human induced pluripotent stem cell (hiPSC)-derived RPE cells, ARPE-19 cells, and adult RPE tissue. (B) hiPSC-derived RPE cells were stained for senescence-associated b-galactosidase (SA-β-gal) activity. Serially passaged hiPSC-derived RPE cells stained strongly with SA-β-gal. (C) The percentages of SA-β-gal-positive RPE cells at passage 0, without passaging, or after serial passaging. (D) Expression of RPE cell markers. (E) Protein expression of the senescence biomarkers p21, p27, and p53 evaluated by Western blotting. P0 = passage 0; NP = non-passaged; PA = serial passaging. *p < 0.05, **p < 0.01.

• 

  Fig. 2 (A) The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of senescent retinal pigment epithelial (RPE) cells treated with anti-vascular endothelial growth factor (VEGF) agents. There was no significant increase in TUNEL-positive cells among senescent RPE cells treated with anti-VEGF agents. (B) Percentage of TUNEL-positive senescent RPE cells. (C) The viability of senescent RPE cells treated with anti-VEGF agents was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (D) The change in pigmentation of senescent RPE cells was evaluated after anti-VEGF treatment. ns = non-specific; sen = senescence; ran = ranibizumab; bev = bevacizumab; afl = aflibercept. *p < 0.05. Bar = 200 µm.

• 

  Fig. 3 (A) Proliferation of senescent retinal pigment epithelial (RPE) cells treated with anti-vascular endothelial growth factor agents were evaluated using the 5-ethynyl-2-deoxyuridine (EdU) assay. Red dots (EdU-positive cells) represent proliferating RPE cells. (B) Percentage of EdU-positive senescent RPE cells. There were no differences in cell proliferation between naïve senescent RPE cells and anti-vascular endothelial growth factor-treated senescent RPE cells. ns = non-specific; sen = senescence; ran = ranibizumab; bev = bevacizumab; afl = aflibercept. Bar = 200 µm.

• 

  Fig. 4 (A) Effects of anti-vascular endothelial growth factor treatment on senescence of retinal pigment epithelial (RPE) cells. (B) Percentage of senescence-associated-β-galactosidase (SA-β-gal) positive senescent RPE cells. Anti-vascular endothelial growth factor treatment did not cause additional senescence of RPE cells. ns = non-specific; sen = senescence; ran = ranibizumab; bev = bevacizumab; afl = aflibercept. Bar = 200 µm.

• 

  Fig. 5 (A) Immunofluorescence staining for zonula occludens-1 (ZO-1). (B) Immunofluorescence staining for RPE65. ZO-1 and RPE65 proteins were uniformly expressed regardless of whether senescent retinal pigment epithelial (RPE) cells were treated with anti-vascular endothelial growth factor agents. (C) Western blot assay for the expression of ZO-1 and RPE65 in senescent RPE cells treated with each of anti-vascular endothelial growth factor agents. sen = senescence; ran = ranibizumab; bev = bevacizumab; afl = aflibercept. Bar = 200 µm.

• 

  Fig. 6 (A) Phagocytosis in senescent retinal pigment epithelial (RPE) cells. Senescent RPE cells were treated with photoreceptor outer segments for 6 hours. Intracellular rhodopsin (red) was detected by immunocytochemistry. The red fluorescence crossing the cell membrane consisted primarily of debris remaining on the cell surface after washing. No difference in the number of rhodopsin positive cells was observed between naïve senescent RPE cells and anti-vascular endothelial growth factor-treated senescent RPE cells. (B) Percentages of rhodopsin-phagocytosing senescent RPE cells. ns = non-specific; sen = senescence; ran = ranibizumab; bev = bevacizumab; afl = aflibercept.

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