Fully Automated Angiogenesis Drug Discovery Assay in Zebrafish

描述（由申请人提供）：斑马鱼Danio rerio已被广泛接受为人类疾病的模型。已经进行了小分子筛选以鉴定血管图案化的修饰剂，但是由于用于全自动处理的工具尚不可用，因此该过程以相对低的通量进行。我们建议开发一种新的，高通量筛选血管生成促进剂的检测方法。该测定将基于使用SideView微孔板来收集在其脉管系统中表达GFP的动物的脉管模式的清晰图像。在第一阶段，我们：1）开发了调节神经丛中血管生成启动子的新方法; 2）开发了定位斑马鱼、识别感兴趣区域并将该区域移动到视场中心的软件;和3）开发了自动化的采集后图像处理软件，用于区分野生型斑马鱼胚胎和神经丛畸形的xxx突变胚胎。在第二阶段，方法、软件和仪器将被完全整合，并且将使用FDA批准的化合物的Prestwick库在模型筛选中验证测定。 公共卫生相关性：第一阶段和第二阶段计划将开发一种全自动工具，用于发现影响血管发育的潜在治疗化合物，这与血管异常等胚胎疾病有关。此外，还将发现促进或抑制新血管生长的化合物，用于治疗包括癌症和湿性黄斑变性在内的多种疾病。该试验将在斑马鱼幼虫中进行，这将使已鉴别化合物在药物开发过程中进行的可能性更高。

DESCRIPTION (provided by applicant): The zebrafish, Danio rerio, has become widely accepted as a model for human disease. Small molecule screens have been performed to identify modifiers of vascular patterning but this process has been performed at relatively low throughput since the tools for fully automated processing are not yet available. We propose to develop a novel, high-throughput screening assay for angiogenesis promoters. The assay would be based on the use of the SideView" microplate to collect clear images of the vascular pattern in animals that express GFP in their vasculature. During Phase I we: 1) developed novel methods to modulate angiogenesis promoters in the plexus; 2) developed software that locates the zebrafish, identifies the region of interest, and moves that region to the center of the field of view; and 3) developed automated, post-acquisition image processing software that distinguishes wild-type zebrafish embryos from xxx mutant embryos with deformities in the plexus. During Phase II the methods, software and instrumentation will be fully integrated and the assay will be validated in a model screen using the Prestwick Library of FDA- approved compounds. PUBLIC HEALTH RELEVANCE: The Phase I and Phase II programs will develop a fully automated tool for discovery of potential therapeutic compounds that affect developing vasculature, which is relevant to embryonic diseases such as vascular anomalies. In addition, compounds that promote or inhibit growth of new blood vessels as a treatment for numerous diseases including cancer and wet macular degeneration will also be discovered. The assay will be performed in zebrafish larvae, which will result in a higher probability that identified compounds, will proceed through the drug development process.