Molecular mechanism of chowidal neovascularization in age-related meler degeneration

年龄相关梅勒变性中乔维达尔新生血管形成的分子机制

基本信息

批准号：
14571659
负责人：
OHNO-MATSUI Kyoko
金额：
$ 2.18万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

We investigated gene expression profiles of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) In differentiated and non-differentiated retinal pigment epithelial (RPE) cells during oxidative stress. Human RPE cells were grown in culture on laminin-coated flasks to obtain differentiated features. Cells cultured on 'plastic were used as non-differentiated controls. After confluence, hydrogen peroxide (H202) was added for 48 h, then, total RNA was extracted and used for RT-PCR and Northern blot analysis. Medium conditioned by RPE was used for ELISA, Western blotting, and in vitro angiogenesis assay. As a result, differehtiated RPE cells expressed significantly higher levels of VEGE protein, as compared to their non-differentiated counterpails. The expression pattern remained consistent even after cellular exposure to H_O_2. Conversely, while elevated levels of PEDF transcript and protein were seen in differentiated RPE cells, compared to non-differentiated dells, a marked decrease at both PEDF mRNA and protein levels was seen after treatment with H_2O_2 Moreover, this decrease in PEDF. expression was dosage dependent In in vitro angiogenesis assay, conditioned medium from differentiated human RPE cells after exposure to H_2O_2 showed a dramatic increase 'in tubular formation and migratory activity of microvascular endothelial cell& These data suggest that, in physiological conditions, a critical balance between PEDF and VEGF exists, and PEDF may counteract the angiogenic potential of VEOF. Under oxidative stress, PEOF decreases disrupting this balance. This equilibrium shift may be significant in promoting a pathological condition of RPE cells and contributing to choroidal neovascularization in age-related macular degeneration.

我们研究了在氧化应激期间分化和非分化的视网膜上皮细胞（RPE）细胞中血管内皮生长因子（VEGF）和色素上皮衍生因子（PEDF）的基因表达谱。人RPE细胞在粘贴蛋白涂层的烧瓶上培养中生长，以获得区分特征。在塑料上培养的细胞用作非差异对照。汇合后，将过氧化氢（H202）添加48小时，然后将总RNA提取并用于RT-PCR和Northern印迹分析。由RPE调节的培养基用于ELISA，蛋白质印迹和体外血管生成测定法。结果，与非分辨率的典型相比，差异的RPE细胞表达的VEGE蛋白水平明显更高。即使在细胞接触H_O_2之后，表达模式仍然保持一致。相反，与非分化的dell相比，在分化的RPE细胞中可以看到PEDF转录物和蛋白质水平升高，但在使用H_2O_2处理后，PEDF mRNA和蛋白质水平显着降低，此外，PEDF的下降。表达取决于体外血管生成测定法，暴露于H_2O_2后分化的人RPE细胞的条件培养基显示出微血管内皮细胞的肾小管形成和迁移活性的急剧增加，这些数据表明，在Pedf和Vegf之间存在着重要的平衡，并且在生理条件下，PEDF和VEVF的势力可能对抗。在氧化应激下，PEOF降低了破坏这种平衡。这种平衡转移可能在促进RPE细胞的病理状况并导致与年龄相关的黄斑变性中有助于新生血管形成。