Optical Tools to Assess the Role of Cardiac Function in the Development of Congenital Heart Defects

项目摘要 我们和其他人已经证明，血流动力学和剪切力的改变会导致先天性心脏缺陷。 (CHDS)，但关于这些力如何影响分子信号的信息仍然有限。研究其影响 血流动力学异常和剪切力的问题变得更加紧迫 血流可能是导致大部分CHD的一个因素，无论最初的触发因素是 环境的或遗传的。尽管我们的团队和其他人最近开发出了非常有用的光学 用于评估血流动力学和剪应力的成像工具(例如，光学相干断层扫描-OCT)，以及 将这些测量与先天性心脏病联系起来，很难将剪切力与受影响的分子联系起来。 小路。我们的团队和其他人在对照和剪切应力扰动下进行了qpr实验。 以了解血流动力学异常如何改变基因表达。但是，这种方法需要整个 胚胎心脏的一次测量，丢失了所有空间和细胞类型信息，特别是在 心内膜层。为了成功评估切应力如何影响整个过程中的分子信号转导 心脏循环，我们需要改进我们的OCT方法，开发3D方法来评估胚胎心脏 基因表达，并创建一条先进的图像处理管道来分析数据和关联区域剪切 应激对基因表达的影响。 这项续订提案将继续我们开发工具的工作，这些工具可以导致更复杂的 了解心脏功能(如血流动力学和电脉冲传导)对心脏的影响 开发，使潜在的治疗方法能够避免或减轻CHD。在这份提案中，我们将重点发展 了解振荡剪切应力(量化为振荡剪切指数-OSI)如何影响基因的工具 表达，并导致先天性心脏病。在我们的初步研究中，我们增加了返流血流量(导致 OSI增加)，以表明OSI的改变会导致较小的心脏垫(瓣膜前体)和 归根结底，是为了CHDS。在我们的两种疾病模型中，返流血流增加和坐垫变小 (胎儿酒精谱系障碍-FASD；胎儿心面部综合征/DiGeorge)和我们的FASD预防 化合物部分地使血流、心脏垫大小正常化，并极大地减少发病率和冠心病。 我们的具体目标包括1)改进我们的OCT系统和剪应力分析，2)发展荧光在 原位杂交(FISH)协议用于测量3D中的基因表达，3)开发图像处理流水线 将基因表达与切应力联系起来，以及4)确定切应力对基因表达的影响。vt.在.的基础上 完成后，我们将有更多关于剪切力如何影响分子表达的信息。使用 有了这些知识，我们就能更好地确定哪些分子途径最容易受到改变的影响 血流动力学，开发更早的检测方法，并潜在地开发策略，以更多地预防CHD 有效地。

Project Summary We and others have shown that altered hemodynamics and shear stress can lead to congenital heart defects (CHDs), but still there is limited information on how these forces affect molecular signaling. Studying the impact of abnormal hemodynamics and shear stress becomes even more urgent when we consider that perturbed blood flow may be a contributing factor to a large percentage of CHDs regardless of whether the initial trigger is environmental or genetic. Although our group and others have recently developed extremely useful optical imaging tools (e.g., optical coherence tomography – OCT) to assess hemodynamics and shear stress, and connected these measurements to CHDs, it has been difficult to link shear stress with the affected molecular pathways. Our group and others have performed qPCR experiments on control and shear-stress perturbed hearts to see how abnormal hemodynamics alters gene expression. However, this approach requires the entire embryonic heart for one measurement, missing all spatial and cell-type information, particularly at the endocardial layer. In order to successfully assess how shear stress affects molecular signaling throughout the looping heart, we need to improve upon our OCT methods, develop 3D methods for assessing embryonic heart gene expression, and create an advanced image processing pipeline to analyze data and relate regional shear stress to gene expression. This renewal proposal will continue our work developing tools that can lead to a more sophisticated understanding of how cardiac function (e.g., hemodynamics and electrical impulse conduction) affects heart development, enabling potential therapies to avoid or mitigate CHDs. In this proposal, we will focus on developing tools to understand how oscillatory shear stress (quantified as oscillatory shear index - OSI) influences gene expression and leads to CHDs. In our preliminary studies, we increased regurgitant blood flow (causing increased OSI) to show that alterations to OSI leads to smaller cardiac cushions (valve precursors) and ultimately, to CHDs. Increased regurgitant blood flow and smaller cushions is present in our two disease models (fetal alcohol spectrum disorders – FASD; velo-cardio-facial syndrome/Digeorge) and our FASD prevention compounds partially normalize blood flow, cardiac cushion size, and greatly reduce morbidity and CHDs. Our specific aims include 1) advance our OCT system and shear stress analysis, 2) develop fluorescence in situ hybridization (FISH) protocols to measure gene expression in 3D, 3) develop an image processing pipeline to relate gene expression to shear stress, and 4) determine the impact of shear stress on gene expression. Upon completion, we will have significantly more information on how shear stress affects molecular expression. With this knowledge, we will be better equipped to determine which molecular pathways are most influenced by altered hemodynamics, to develop earlier detection methods and potentially develop strategies to prevent CHDs more effectively.