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

描述（由申请人提供）：血管内皮屏障的调节是一个协调的信号传导过程，控制氧气和营养物质与周围组织的交换。屏障完整性或功能失调涉及一系列病理学，包括代谢疾病如糖尿病（影响8.3%的美国人群）和心血管疾病如动脉粥样硬化（影响25%的美国人群）。屏障的调节由小的气体反应性信号分子（例如一氧化氮和超氧化物）控制，这些分子已被证明在细胞中具有足够的选择性和灵敏度来检测和定量具有挑战性，更重要的是在复杂组织和活动物中。成像提供了重要的洞察调节的屏障和相关的 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没有足够的特异性或灵敏度来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，这阻止了分子信号传导和血管生理学的同时相关测量。在这个提议中，我们开发了一类新的荧光分子生物传感器染料，该染料联合收割机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遗传编码的亚细胞靶向，这些新的生物传感器将允许在特定的亚细胞位置检测特定细胞中的Ca（II）、活性氧（ROS）和活性氮（RNS）。这些新型靶向荧光生物传感器通过将Ca、ROS或RNS的敏感光学传感器与荧光信号传导部分（FRET受体）连接在一起来构建，所述荧光信号传导部分在结合到遗传编码的受体（称为荧光激活蛋白（FAP））时被激活。FAP结合的传感器能够报告（荧光信号）感兴趣部位的感测生理学。任何未与FAP靶标结合的生物传感器都不能产生荧光信号，并且没有使图像或数据分析复杂化的背景或非特异性荧光。靶向生物传感器染料将被优化以在培养的内皮细胞和活的斑马鱼中工作。使用组织特异性Cre重组酶表达，将产生在特定细胞的亚细胞位置表达FAP的转基因斑马鱼。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。该项目是三个主要研究人员在匹兹堡大学和梅隆大学的独特专业知识的密切合作。Bruchez博士是开发用于生物检测的多发色团结构的专家，并设计了使用荧光团活化蛋白进行生物传感的混合指示剂; St.克鲁瓦博士是内皮细胞生物学RNS/ROS信号传导和内皮功能调节的专家，更具体地说是体外和体内内皮成像的专家。Waggoner博士是环境敏感染料设计方面的专家，也是荧光激活蛋白技术的原始开发者。

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.