Novel High Content Vascular Patterning Assay(RMI)

描述(申请人提供)：血管生成，新血管的生长，发生在一系列不同的人类疾病中。尽管抗血管生成药物仍然是一个巨大的药物市场，但基于高含量、高通量的基于细胞的血管生成抑制剂发现方法仍有待开发。我们相信，开发新的检测方法作为筛选工具，以发现靶向内皮细胞分化的抑制剂，可以被证明是识别新类别抗血管生成药物的有效手段。在这里，我们建议开发一种高含量的血管模式分析，很可能被用于高通量筛选(HTS)。我们计划通过将高通量筛选能力与三维(3-D)基质中内皮细胞图案的多参数光学成像相结合，来构建一个强大的高内容筛选平台的原型。我们将通过以下目标实现我们实现这些目标的创新方法。在具体目标1中，我们将建立并验证三维内皮细胞图案化分析(3-D ECPA)。我们将通过开发方法在微载体珠子上生成高度健壮的多细胞内皮细胞球体，并在384孔光学筛选板中将球体排列在3-D胶原基质中来实现这一点。此外，这些检测将在低氧环境下进行。在具体目标2中，我们计划开发和验证一个用于3-D ECPA的高内容(HC)图像分析平台。这将通过开发软件来实现，该软件可以识别3-D基质中内皮细胞图案的新特征，并应用自动化分析来获得形态指纹。最后，我们将通过筛选一组不同的天然产物来验证3-DECPA和图像分析软件在高温超导应用中的有效性。我们相信，这个HC-HTS平台将提供独特的机会来筛选大量的小分子血管生成抑制剂，并帮助药物开发。

DESCRIPTION (provided by applicant): Angiogenesis, the growth of new blood vessels, occurs in a diverse array of human diseases. Even though anti-angiogenesis remains a large market for Pharmaceuticals, high content high throughput cell based assays for angiogenesis inhibitor discovery remain to be developed for this application. It is our belief that developing new assays as screening tools for discovery of inhibitors that target endothelial cell differentiation can prove to be an efficient means of identifying new classes of anti-angiogenic drugs. Here we propose to develop a high content vascular patterning assay that can likely be adopted for high throughput screening (HTS). We plan to build a prototype of a powerful high content screening platform by interfacing high throughput screening capability with multiparameter optical imaging of endothelial cell patterning in three dimensional (3-D) matrices. Our innovative approach to achieve these goals will be accomplished through the following aims. In Specific aim 1 we will develop and validate a 3-D endothelial cell patterning assay (3-D ECPA). We will accomplish this by developing methods to generate highly robust multicellular endothelial cell spheroids on microcarrier beads and array the spheroids in 3-D collagen matrix in 384 well optical screening plates. In addition, these assays will be performed under hypoxic environment. In Specific Aim 2 we plan to develop and validate a high content (HC) image analysis platform for use with 3-D ECPA. This will be accomplished by developing software that identifies novel features of endothelial cell patterning in 3-D matrix and apply automated analysis to obtain morphogenic fingerprints. Finally, we will validate the 3-DECPA and image analysis software for HTS application by screening a diversity set of natural products. We believe that this HC-HTS platform will offer unique opportunities to screen vast numbers of small molecule inhibitors of angiogenesis and assist in drug discovery.