Vascular endothelial growth factor as an autocrine survival factor for retinal pigment epithelial cells under oxidative stress via the VEGF-R2/PI3K/Akt.
OBJECTIVE
Vascular endothelial cell growth factor (VEGF) is strongly induced by oxidative stress in retinal pigment epithelial (RPE) cells, and VEGF-A is a survival factor for various cell types. This study was conducted to determine whether the autocrine VEGF signaling pathway in RPE cells is involved in the mechanism of adaptive response to oxidative stress.


METHODS
ARPE-19 cells were treated with hydrogen peroxide, and cell death was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Survival analysis was performed with pretreatment of VEGF-A-neutralizing antibodies, VEGF receptor tyrosine kinase inhibitor (SU5416), or VEGF-A receptor-neutralizing antibodies (anti-VEGF-R1 and anti-VEGF-R2). The expression of VEGF-A, -R1, -R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR or Western blot analysis. Phosphorylation of VEGF-R2 was detected with immunoprecipitation and immunoblot analysis.


RESULTS
Hydrogen peroxide-induced cell death was promoted by pretreatment with VEGF-A and anti-VEGF-R2-neutralizing antibodies, but not with anti-VEGF-R1-neutralizing antibody. Phosphorylation of VEGF-R2 in RPE cells was induced by hydrogen peroxide, and pretreatment with anti-VEGF-A-neutralizing antibody inhibited phosphorylation. Phosphorylation of Akt under oxidative stress was abrogated by pretreatment with neutralizing antibodies against either VEGF-A or SU5416.


CONCLUSIONS
Autocrine VEGF-A enhanced RPE cell survival under oxidative stress; the autocrine VEGF-A/VEGF-R2/PI3K/Akt pathway is involved. Neutralization of VEGF-A signaling, as in eyes with age-related macular degeneration, may influence RPE cell survival.
Byeon
Suk Ho
SH
Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. shbyeon@yuhs.ac
Lee
Sung Chul
SC
Choi
Soo Hyun
SH
Lee
Hyung-Keun
HK
Lee
Joon H
JH
Chu
Young Kwang
YK
Kwon
Oh Woong
OW
D017209:Apoptosis; D019898:Autocrine Communication / Q000502:physiology*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002470:Cell Survival; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051058:Oncogene Protein v-akt / Q000378:metabolism; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D051058:Oncogene Protein v-akt