Novel Modular Vascular Patterning Assay for HTS

描述(申请人提供)：人体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，微血管动态平衡是人类健康的重要组成部分；它在炎症和血管生成刺激下的不适当激活可能成为助长癌症生长和扩散的致病成分，并导致衰弱的关节炎、老年性黄斑变性和与糖尿病等基础疾病相关的多器官衰竭。另一方面，血管生成受损也同样具有致病性，通过减缓伤口愈合并导致心脏病和中风来折磨受害者。总体而言，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主要负担需要通过更协调一致的药物发现努力来解决。模拟血管生成疾病过程的生物测试可以帮助药物发现和疾病靶点确定的过程。然而，目前很少有检测方法代表病变微血管系统的复杂性，因为它们通常集中在一种致病机制/途径上。考虑到这一点，我们建议继续开发一种高含量高通量筛选(HC-HTS)血管模式分析，我们最近通过之前资助的NIH路线图倡议R21拨款将其验证为药物筛选工具。在这份R01提案中，我们计划扩大三维内皮细胞发芽试验(3D-ECSA)的规模和范围，以促进其被HTS范式采用。我们的创新方法带来了1)现代自动化机器人系统，使我们能够提高球体的效率和标准化生产，2)高含量图像分析软件，用于高温超导条件下的3D-ECSA，以及3)开发一个中试规模的化学库，专注于免疫蛋白酶体，作为验证3D-ECSA的化学使能工具。这些目标的成功实现不仅将为大规模生物学提供有价值的工具，而且将有助于推动技术进步，为挽救生命的治疗寻找新的蛋白质功能化学探针和药物先导。

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.