Angiogenin调节视网膜色素上皮细胞EMT参与湿性年龄相关性黄斑变性的机制研究

Choroidal neovascularization (CNV) and subretinal fibrosis are the major causes of vision loss in the progression of age-related macular degeneration (AMD). Epithelial-to-mesenchymal transition (EMT）of retinal pigment epithelium (RPE) is the most important pathological process in the formation of subretinal fibrosis. By using the mouse model of laser-CNV to mimic the wet form of human AMD, we found an elevated expression level of Angiogenin (ANG) and a decreased CNV area in Ang1-/- mice in the previous study. These results indicate an important role of ANG in wet AMD. The study in vitro showed ANG could also regulate the expression of Vimentin, an EMT marker, and contribute to the EMT of RPE, besides of the promotion of tube formation in human retinal microvascular endothelial cells. Based on the previous work, this project aims to demonstrate the function of ANG in CNV formation and the EMT of RPE by using Ang1-/- mice and laser-CNV model. Next, we will verify the role of ANG in EMT of RPE and explore the possible molecular mechanisms in vitro. Finally, we will identify the outcomes of anti-ANG agents (Ang1 antibodies or blocking peptide) in treating wet AMD by intravitreous injection. In our view, the project may reveal the new insights of wet AMD and provide evidence of new therapeutic targets for wet AMD from both aspects of CNV and subretinal fibrosis.

脉络膜新生血管（CNV）和视网膜下纤维化是年龄相关性黄斑变性（AMD）视力丧失的主要原因。视网膜色素上皮细胞（RPE）间充质转分化（EMT）是视网膜下纤维化的重要病理过程。利用激光小鼠CNV模型模拟人湿性AMD发病过程，我们发现CNV病灶中血管生成素（Angiogenin，ANG）表达升高，Ang1基因敲除小鼠CNV区域面积减小。体外研究表明，除促血管新生外，ANG还可调节RPE中EMT标志物Vimentin表达，参与EMT。为此，本项目拟利用Ang1基因敲除小鼠、激光CNV模型明确ANG在CNV形成及RPE细胞EMT中的作用；利用体外细胞模型探究ANG调控RPE细胞EMT的分子机制；最后通过小鼠玻璃体腔注射Ang1抗体或抑制肽阻断Ang1与受体的结合，评价靶向ANG治疗湿性AMD的可行性。本项目研究将有助于深入了解湿性AMD的发病机制，并为湿性AMD血管新生和纤维化两方面的治疗提供靶点。

脉络膜新生血管（CNV）和视网膜下纤维化是年龄相关性黄斑变性（AMD）视力丧失的主要原因。前期体外研究中我们发现，除促血管新生外，ANG还可调节RPE中EMT标志物Vimentin表达，参与上皮细胞间充质转分化（EMT）。本研究为探索ANG调节视网膜色素上皮细胞EMT参与AMD的机制，首先，在动物水平，我们通过传统基因敲除技术，成功构建了并繁殖了Ang1 敲除的小鼠（Ang1-/-），利用WT小鼠和Ang1-/-小鼠施行视网膜激光以模拟湿性AMD。分别在施行激光术后3天和7天，取小鼠RPE/脉络膜复合物提取RNA和蛋白，检测小鼠RPE/脉络膜复合物中间质细胞分子标志（Vimentin）、上皮细胞分子标志物（E-cadherin）以及反应纤维化的α-SMA的表达。我们发现WT激光小鼠较对照组E-cadherin表达下降，Vimentin、α-SMA表达升高；而Ang1-/-激光小鼠较WT激光小鼠E-cadherin表达上升，Vimentin、α-SMA表达下降。由此，我们可以得出结论ANG可以促进小鼠RPE细胞EMT。在细胞水平，我们利用体外培养的人视网膜色素上皮细胞系ARPE-19，发现外源性添加ANG后，ARPE-19细胞Vimentin的表达上升、E-cadherin的表达下降，细胞增殖水平、迁移水平提高。为进一步探索机制，我们发现ANG可以激活JNK信号转导通路，而预加入JNK信号通路抑制剂SP600125可以使ANG诱导的ARPE-19细胞Vimentin的表达上升得到抑制、E-cadherin的表达下降明显回升。另外，预加入ANG/Plexin B2结合竞争性抑制肽，可以抑制p-c-jun的表达水平。上述结果提示ANG通过活化JNK信号通路参与RPE细胞EMT。本项目研究将有助于推动ANG抗体或结合竞争性抑制肽从血管新生和纤维化两方面应用于湿性AMD的治疗。