Department of Ophthalmology, Shanghai Jiaotong University affiliated Shanghai First People's Hospital, Shanghai, China.
Curr Eye Res. 2011 Apr;36(4):379-85. doi: 10.3109/02713683.2010.537427. Epub 2011 Feb 24.
Native and oxidized (OX) low-density lipoprotein (LDL) may contribute to the pathogenesis of age related macular degeneration (AMD). In this study, we investigated the effects of lipoproteins, including LDL and ox-LDL, on cell viability, apoptosis, and vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) expression in cultured human retinal pigment epithelial (RPE) cells.
ARPE-19 cells were incubated with 10-100 mg/ml n-LDL, ox-LDL for 24 hr. Cell viability was assessed using the Cell Titer 96 Aqueous One Solution cell proliferation assay. The apoptosis of RPE was measured with TUNEL. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF and PEDF mRNA in RPE cells. The expression of VEGF and PEDF protein was measured by western blotting. To examine the role of MAPK signal transduction in LDL- and OX-LDL-induced VEGF and PEDF protein expression, ARPE-19 cells were pretreated with one of several MAPK inhibitors for 2 hr and then incubated with native LDL or OX-LDL for 24 hr. One-way analysis of variance was used to compare the differences.
OX-LDL treatment decreased ARPE-19 cell viability in a dose-dependent manner, whereas native LDL had no effect. Incubation of ARPE-19 cells with 10 mg/mL OX-LDL induced marked apoptosis, compared with untreated control cells. OX-LDL also increased VEGF expression and decreased PEDF expression, whereas native LDL had no significant effect. The VEGF-to-PEDF ratio was elevated after OX-LDL treatment. OX-LDL-induced VEGF protein synthesis was partly abolished by inhibiting p38 and JNK, while inhibiting ERK did not show a significant effect.
OX-LDL treatment induced cellular changes in ARPE-19 cells that appeared to reflect pathogenic events in neovascular AMD, providing potential insight into the roles of OX-LDL in human RPE cells and its potential role in the pathogenesis of AMD.
天然和氧化（OX）低密度脂蛋白（LDL）可能有助于年龄相关性黄斑变性（AMD）的发病机制。在这项研究中，我们研究了脂蛋白，包括 LDL 和 ox-LDL，对培养的人视网膜色素上皮（RPE）细胞活力、凋亡以及血管内皮生长因子（VEGF）和色素上皮衍生因子（PEDF）表达的影响。
用 10-100mg/ml 的 n-LDL、ox-LDL 孵育 ARPE-19 细胞 24 小时。用 Cell Titer 96 Aqueous One Solution 细胞增殖检测试剂盒评估细胞活力。用 TUNEL 法检测 RPE 细胞凋亡。用逆转录聚合酶链反应（RT-PCR）检测 RPE 细胞中 VEGF 和 PEDF mRNA 的水平。用 Western blot 法检测 VEGF 和 PEDF 蛋白的表达。为了研究 MAPK 信号转导在 LDL 和 OX-LDL 诱导的 VEGF 和 PEDF 蛋白表达中的作用，ARPE-19 细胞先用几种 MAPK 抑制剂预处理 2 小时，然后再用天然 LDL 或 OX-LDL 孵育 24 小时。用单因素方差分析比较差异。
OX-LDL 处理呈剂量依赖性降低 ARPE-19 细胞活力，而天然 LDL 则无此作用。与未处理的对照细胞相比，10mg/ml OX-LDL 孵育诱导 ARPE-19 细胞明显凋亡。OX-LDL 还增加了 VEGF 表达，降低了 PEDF 表达，而天然 LDL 则无显著影响。OX-LDL 处理后 VEGF 与 PEDF 的比值升高。抑制 p38 和 JNK 部分阻断了 OX-LDL 诱导的 VEGF 蛋白合成，而抑制 ERK 则没有显著作用。
OX-LDL 处理诱导 ARPE-19 细胞发生细胞变化，似乎反映了新生血管性 AMD 的发病机制事件，为 OX-LDL 在人 RPE 细胞中的作用及其在 AMD 发病机制中的潜在作用提供了潜在的见解。
