Targeted fluorescent indicators for endothelial physiology: Ca(II), ROS, NO

描述(申请人提供)：血管内皮细胞屏障的调节是一个协调的信号过程，控制氧气和营养物质与周围组织的交换。屏障完整性或功能的失调涉及一系列病理，包括代谢紊乱，如糖尿病(影响8.3%的美国人口)和心血管疾病，如动脉粥样硬化(影响25%的美国人口)。屏障的调节由小的气态反应性信号分子(如一氧化氮和超氧化物)控制，事实证明，在细胞中，更重要的是在复杂的组织和活的动物中，具有足够的选择性和灵敏度来检测和定量具有挑战性的信号分子。成像提供了对屏障和相关的调节的重要洞察 与疾病和疾病治疗相关的生理变化。然而，与屏障相关的信号分子的成像继续构成重大挑战，因为目前可用的荧光生物传感器不够具体或足够灵敏，无法直接报告分析物的浓度和位置。基于染料的荧光探针和荧光蛋白传感器都受到限制，无法同时进行分子信号和血管生理学的相关测量。在这个建议中，我们开发了一类新的荧光分子生物传感器染料，它结合了指示染料的优点和遗传编码的特异性。通过使用组织特异性表达和基因编码的亚细胞靶向，这些新的生物传感器将能够在特定的亚细胞位置检测特定细胞中的钙(II)、活性氧(ROS)和活性氮(RNS)。这些新型的靶向荧光生物传感器是通过将敏感的钙、ROS或RNS的光学传感器与荧光信号部分(FRET受体)连接在一起而构建的，FRET受体在与称为氟原激活蛋白(FAP)的遗传编码受体结合时被激活。FAP结合的传感器能够报告(荧光信号)感兴趣部位的传感的生理学。任何未与FAP靶标结合的生物传感器都不能产生荧光信号，也没有背景或非特定的荧光使图像或数据分析复杂化。目标生物传感器染料将进行优化，以在培养的内皮细胞和活的斑马鱼中发挥作用。将产生转基因斑马鱼，利用组织特异性Cre重组酶表达在特定细胞的亚细胞位置表达FAP。我们将在斑马鱼中使用这些传感器来评估Ca(II)、ROS和NO信号、血流和屏障功能之间的相关性。这个项目是由卡内基梅隆大学和匹兹堡大学的三名具有不同专业知识的首席研究人员密切合作的。Bruchez博士是开发用于生物检测的多色团结构的专家，并使用荧光素激活蛋白设计了用于生物传感的混合指示器；St.Croix博士是内皮细胞生物学、RNS/ROS信号和内皮功能调节方面的专家，更具体地说，是体外和体内内皮成像方面的专家。瓦格纳博士是环境敏感染料设计方面的专家，也是氟原激活蛋白技术的最初开发者。

DESCRIPTION (provided by applicant): The regulation of the endothelial barrier in blood vessels is a coordinated signaling process that controls the exchange of oxygen and nutrients with the surrounding tissues. Dysregulation of barrier integrity or function is implicated in a range of pathologies, including metabolic disorders such as diabetes (affecting 8.3% of the US population) and cardiovascular disorders such as atherosclerosis (affecting 25% of the US population). Regulation of the barrier is controlled by small gaseous reactive signaling molecules (e.g. nitric oxide and superoxide) that have proven challenging to detect and quantify with adequate selectivity and sensitivity in cells and more importantly in complex tissues and living animals. Imaging has provided significant insight into regulation of the barrier and related physiologic changes that correlate with disease and disease treatment. Yet imaging of signaling molecules associated with the barrier continues to pose significant challenges because the currently available fluorescent biosensors are not sufficiently specific or sensitive to directly report the concentrations and locations of the analytes. Both dye based fluorescent probes and fluorescent protein sensors suffer from limitations that prevent simultaneous correlative measurements of molecular signaling and vascular physiology. In this proposal, we develop a new class of fluorescent molecular biosensor dyes that combine the advantages of indicator dyes with the specificity of genetic encoding. By using tissue specific expression and genetically encoded subcellular targeting, these new biosensors will allow detection of Ca(II), reactive oxygen species (ROS), and reactive nitrogen species (RNS) in specific cells, at specific subcellular locations. These novel targeted fluorescent biosensors are constructed by linking together a sensitive optical sensor of Ca, ROS, or RNS with a fluorescent signaling moiety (FRET acceptor) that is activated upon binding to a genetically encoded receptor, called a fluorogen activating protein (FAP). FAP-bound sensor is able to report (fluorescence signal) the physiology of the sensing at the site of interest. Any biosensors that are not bound to the FAP target are incapable of producing a fluorescence signal and there is no background or non-specific fluorescence to complicate images or analysis of the data. The targeted biosensor dyes will be optimized to work in both cultured endothelial cells and living zebrafish. Transgenic zebrafish will be generated that express the FAP at subcellular locations in specific cells using tissue specific Cre-recombinase expression. We will use these sensors in zebrafish to assess the correlation between Ca(II), ROS and NO signaling, blood flow and barrier function. This project is a close collaboration of three Principal Investigators with distinct expertise at Carnege Mellon University and University of Pittsburgh. Dr. Bruchez is an expert on the development of multichromophore structures for biological detection, and designed the hybrid indicators for biosensing using the fluorogen activating proteins; Dr. St. Croix is an expert on endothelial cell biology RNS/ROS signaling and regulation of endothelial function and more specifically the imaging of endothelium in vitro and in vivo. Dr. Waggoner is an expert in the design of environmentally sensitive dyes, and original developer of the fluorogen activating protein technology.