Fully Automated Angiogenesis Drug Discovery Assay in Zebrafish

描述(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来识别血管图案的修饰剂，但由于用于全自动处理的工具尚不可用，该过程以相对较低的吞吐量进行。我们建议开发一种新的、高通量的血管生成促进剂的筛选方法。该分析将基于SideView“微板”的使用，以收集在其血管系统中表达GFP的动物的血管图案的清晰图像。在第一阶段，我们：1)开发了调控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野中心的软件；以及3)开发了自动化的采集后图像处理软件，用于区分神经丛中具有畸形的野生型斑马鱼胚胎和xxx突变胚胎。在第二阶段，方法、软件和仪器将完全集成，并将使用FDA批准的化合物的Prestwick库在模型屏幕上验证该分析。 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，用于发现影响发育中的血管系统的潜在治疗化合物，这与胚胎疾病有关，如血管异常。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。该检测将在斑马鱼幼体中进行，这将导致已识别的化合物更有可能在药物开发过程中进行。

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.