New Endothelial Cell-Based Assay to Assess Variability of Nitric Oxide Production in Humans

项目总结/摘要 心血管疾病（CVD）是美国和世界范围内的主要死亡原因。2009年，更多 超过8000万美国人患有CVD，相关费用为3130亿美元。CVD的发展和 其他慢性循环系统疾病与接触环境毒物有关。的 有毒物质可以改变血管功能的关键调节剂-一氧化氮（NO）的生物利用度， 通过内皮细胞。血管内皮细胞NO生成的障碍可能有助于发展 慢性循环系统疾病然而，这并不能解释为什么有些人会患上这种疾病， 其他人则没有。因此，国家环境健康科学研究所领导的努力， 了解个体易感性对复杂疾病（如CVD）的影响。为了帮助这些努力， Creative Scientist，Inc. (CSI)正在开发先进的工具， 血管对环境毒物的敏感性CSI采用分离的内皮集落形成细胞 ECFC是参与胚胎和成体血管发生的独特的原代细胞。ECFC可以是 从血液中分离并冷冻保存。ECFC的捐助方特异性和强劲的增长潜力使得 检测来自任何人的细胞中的数万种化学物质。CSI开发了 专有方案和细胞生长培养基补充，以从少量血液中分离ECFC。我们 本发明的目的是使使用新颖且高度特异性的NO试剂和高含量的NO检测服务商业化。 成像技术在目标1中，我们将确定我们的新的NO特异性荧光的灵敏度和选择性， 探针这一目标的实现将证明我们的试剂用于ECFC中NO测量的实用性。在Aim中 2，我们将在整个细胞培养过程中确定不同ECFC谱系中NO测量的再现性。 目标2的完成将确立ECFC用于鉴定NO抑制剂的实用性。在第二阶段，我们将 测试分析的能力，以检测变异性的反应，1,000毒物在收集的ECFC衍生 500名捐赠者。我们的最终目标是为监管机构提供商业服务， 制药公司旨在评估环境毒物和药物的潜在血管毒性， 在人群中的候选人，特别是试图确定敏感和耐药的个人和/或 人口分组。

PROJECT SUMMARY/ABSTRACT Cardiovascular diseases (CVD) are the leading cause of death in the US and worldwide. In 2009 alone, more than 80 million Americans suffered from CVD with associated cost $313 billion. The development of CVD and other chronic circulatory disorders has been associated with exposure to environmental toxicants. The toxicants can alter bioavailability of a key regulator of vascular function – nitric oxide (NO), which is produced by endothelial cells. The impairment of vascular endothelial NO production can contribute to the development of chronic circulatory disorders. However, this does not explain why some individuals develop the disease and others do not. Accordingly, the National Institute of Environmental Health Sciences leads the efforts to understand the contribution of individual susceptibility to complex diseases, such as CVD. To aid these efforts, Creative Scientist, Inc. (CSI) is developing advanced tools enabling the generation of data on individual vascular susceptibility to environmental toxicants. CSI employs isolated Endothelial Colony-Forming Cells (ECFC), which are unique primary cells involved in both embryonic and adult vasculogenesis. ECFCs can be isolated from blood and cryopreserved. Donor-specific nature and robust growth potential of ECFCs allow for examining tens of thousands of chemicals in cells derived from any number of people. CSI has developed proprietary protocols and cell growth media supplement to isolate ECFCs from small volumes of blood. Our goal is to commercialize an NO testing service using novel and highly specific NO reagent and a high content imaging technique. In Aim 1, we will determine sensitivity and selectivity of our novel NO-specific fluorescent probe. Completion of this Aim will demonstrate the utility of our reagent for NO measurement in ECFCs. In Aim 2, we will determine reproducibility of NO measurement in different ECFC lineages throughout the cell culture. Completion of Aim 2 will establish the utility of ECFC for an identification of NO inhibitors. In Phase II, we will test assay’s capability to detect variability of responses to 1,000 toxicants in a collection of ECFCs derived from up to 500 donors. Our ultimate goal is provide a commercial service to regulatory bodies and pharmaceutical companies aiming to evaluate potential vascular toxicity of environmental toxicants and drug candidates in a population and, in particular, trying to identify both sensitive and resistant individuals and/or population sub-groups.