血管内皮生长因子作为一种自分泌生存因子，通过 VEGF-R2/PI3K/Akt 途径在氧化应激下对视网膜色素上皮细胞起作用。
Vascular endothelial growth factor as an autocrine survival factor for retinal pigment epithelial cells under oxidative stress via the VEGF-R2/PI3K/Akt.
机构信息
Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
出版信息
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1190-7. doi: 10.1167/iovs.09-4144. Epub 2009 Oct 15.
PURPOSE
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.
目的
血管内皮生长因子（VEGF）在视网膜色素上皮（RPE）细胞中受到氧化应激的强烈诱导，VEGF-A 是多种细胞类型的存活因子。本研究旨在确定 RPE 细胞中的自分泌 VEGF 信号通路是否参与了对氧化应激的适应性反应机制。
方法
用过氧化氢处理 ARPE-19 细胞，并用 Annexin V-异硫氰酸荧光素通过流式细胞术测量细胞死亡。用 VEGF-A 中和抗体、VEGF 受体酪氨酸激酶抑制剂（SU5416）或 VEGF-A 受体中和抗体（抗 VEGF-R1 和抗 VEGF-R2）预处理后进行生存分析。通过半定量 RT-PCR 或 Western blot 分析测定 VEGF-A、-R1、-R2 和可溶性 VEGF-R1 的表达。用免疫沉淀和免疫印迹分析检测 VEGF-R2 的磷酸化。
结果
用 VEGF-A 和抗 VEGF-R2 中和抗体预处理可促进过氧化氢诱导的细胞死亡，但用抗 VEGF-R1 中和抗体预处理则不行。过氧化氢诱导 RPE 细胞中 VEGF-R2 的磷酸化，用 VEGF-A 中和抗体预处理则抑制了磷酸化。用中和抗体（VEGF-A 或 SU5416）预处理可阻断氧化应激下 Akt 的磷酸化。
结论
自分泌 VEGF-A 增强了 RPE 细胞在氧化应激下的存活；自分泌 VEGF-A/VEGF-R2/PI3K/Akt 通路参与其中。VEGF 信号转导的中和，如在年龄相关性黄斑变性的眼中，可能会影响 RPE 细胞的存活。
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