Novel Modular Vascular Patterning Assay for HTS

描述（由申请人提供）：人类血管内衬的血管内皮细胞是参与多种人类疾病的炎症和增殖反应的主要部位之一。因此，微血管稳态是人类健康的重要组成部分;其响应于炎症和血管生成刺激的不适当激活可成为致病组分，促进癌症的生长和扩散，并导致衰弱性关节炎、年龄相关性黄斑变性和与潜在疾病如糖尿病相关的多器官衰竭。另一方面，受损的血管生成也同样具有致病性，并通过减缓伤口愈合和促成心脏病和中风来折磨其受害者。总的来说，考虑到血管生成信号系统的复杂性，这些由血管生长失调引起的人类健康的重大负担需要通过药物发现方面更加协调一致的努力来解决。模拟血管生成性疾病过程的生物测定可以辅助药物发现和疾病靶点鉴定的过程。然而，目前很少有分析代表患病微血管的复杂性，因为它们通常集中在一种致病机制/途径上。考虑到这一点，我们建议继续开发高含量高通量筛选（HC-HTS）血管图案分析，我们最近通过先前资助的NIH路线图倡议R21赠款验证了该方法作为药物筛选工具。在R 01提案中，我们计划扩展三维内皮细胞发芽试验（3D-ECSA）的规模和范围，以促进其在HTS范例中的采用。我们的创新方法带来了1）现代自动化机器人系统，使我们能够提高效率并标准化球状体的生产，2）在HTS条件下与3D-ECSA一起使用的高内容图像分析软件，以及3）开发中试规模的化学库，重点是免疫蛋白体作为验证3D-ECSA的化学使能工具。这些目标的成功实现不仅为我们提供了大规模生物学的宝贵工具，而且将有助于推动技术进步，以确定蛋白质功能的新化学探针和挽救生命的药物先导物。

DESCRIPTION (provided by applicant): The vascular endothelial cells lining blood vessels in humans are one of the principle sites that become involved in inflammatory and proliferative responses to a diverse array of human diseases. Microvascular homeostasis is thus a vital component of human health; its inappropriate activation in response to inflammatory and angiogenic stimuli can become a pathogenic component fueling the growth and spread of cancers, and contributing to debilitating arthritis, age-related macular degeneration and multiple organ failure associated with underlying diseases such as diabetes. On the other hand, impaired angiogenesis is also equally pathogenic, and afflicts its victims by slowing down wound healing and contributing to heart diseases and stroke. Collectively, given the complexity of the angiogenesis signaling system, these major burdens of human health that arise from dysregulation of blood vessel growth need to be addressed by a more concerted effort in drug discovery. Biological assays that model the processes of angiogenic diseases can assist the process of drug discovery and disease target identification. However, currently few assays represent the complexity of the diseased microvasculature as they typically focus on one pathogenic mechanism/pathway. With this in mind, we propose to continue the development of a high content high throughput screening (HC-HTS) vascular patterning assay, which we have recently validated as drug screening tool through a previously funded NIH Roadmap Initiative R21 grant. In this R01 proposal, we plan to extend the scale and scope of the three dimensional endothelial cell sprouting assay (3D-ECSA) to promote its adoption for the HTS paradigm. Our innovative approaches bring in 1) modern automated robotic systems that allow us to improve efficiency and standardize production of spheroids, 2) high content image analysis software to use with 3D-ECSA under HTS conditions, and 3) development of a pilot scale chemical library focused on the immunoproteosome as a chemical enabling tool towards validation of the 3D-ECSA. The successful accomplishments of these goals will not only afford us a valuable tool for large scale biology, but will help bring forward a technology advancement to identify new classes of chemical probes of protein function and drug leads for life saving therapeutics.