Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Curr Eye Res. 2012 Jun;37(6):500-7. doi: 10.3109/02713683.2012.663855.
Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A(2) group VIA (iPLA(2)-VIA) to be a potential regulator of cell proliferation and migration, and evidence show abundant expression of iPLA(2)-VIA in RPE cells. The aim of the present study was to evaluate the potential role of iPLA(2)-VIA in VEGF-induced proliferation and migration of RPE cells.
The human RPE cell line, ARPE-19, was used in all assays. To explore the role of iPLA(2)-VIA in VEGF-induced RPE proliferation and migration, iPLA(2)-VIA inhibition by the iPLA(2)-VIA specific inhibitor, bromoenol lactone, was done. RPE cell proliferation and migration were evaluated by measurements of incorporated radioactive thymidine in DNA and by a Boyden chamber technique, respectively. A luciferase assay monitored the VEGF-induced iPLA(2)-VIA transcriptional activity. Western blot analysis and an activity assay were used to detect the protein levels and activity of iPLA(2)-VIA respectively after treatment with VEGF.
RPE cells treated with VEGF showed significant increased proliferation and migration. Furthermore, inhibition of iPLA(2)-VIA significantly reduced the spontaneous proliferation and migration as well as the VEGF-induced proliferation and migration. Finally, inhibition of iPLA(2)-VIA reduced the VEGF-induced iPLA(2)-VIA-activity, -protein level, and -promoter activity.
A significant interaction between VEGF and iPLA(2)-VIA in the regulation of RPE cells appears to be relevant in elucidating the exact mechanisms of action in the proliferative and migratory phenotype of RPE cells in AMD.
抑制血管内皮生长因子（VEGF）是减少年龄相关性黄斑变性（AMD）中视网膜色素上皮（RPE）增殖和迁移的重要治疗方法。此外，先前的研究表明钙非依赖性磷脂酶 A2 组 VIA（iPLA2-VIA）是细胞增殖和迁移的潜在调节剂，并且有证据表明 iPLA2-VIA 在 RPE 细胞中大量表达。本研究旨在评估 iPLA2-VIA 在 VEGF 诱导的 RPE 细胞增殖和迁移中的潜在作用。
所有实验均使用人 RPE 细胞系 ARPE-19。为了探讨 iPLA2-VIA 在 VEGF 诱导的 RPE 增殖和迁移中的作用，使用 iPLA2-VIA 特异性抑制剂溴烯内酯抑制 iPLA2-VIA。通过测量 DNA 中掺入的放射性胸腺嘧啶核苷来评估 RPE 细胞增殖，通过 Boyden 室技术评估迁移。通过荧光素酶测定监测 VEGF 诱导的 iPLA2-VIA 转录活性。Western blot 分析和活性测定分别用于检测 VEGF 处理后 iPLA2-VIA 的蛋白水平和活性。
用 VEGF 处理的 RPE 细胞显示出明显增加的增殖和迁移。此外，抑制 iPLA2-VIA 显著降低了自发增殖和迁移以及 VEGF 诱导的增殖和迁移。最后，抑制 iPLA2-VIA 降低了 VEGF 诱导的 iPLA2-VIA 活性、蛋白水平和启动子活性。
VEGF 和 iPLA2-VIA 之间的显著相互作用似乎与阐明 AMD 中 RPE 细胞增殖和迁移表型的确切作用机制有关。
