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

项目摘要 我们和其他人已经表明，改变血流动力学和剪切应力可导致先天性心脏缺陷 （CHDs），但关于这些力如何影响分子信号传导的信息仍然有限。研究影响 当我们考虑到扰动的血液， 无论最初的触发因素是否存在，流量可能是导致很大一部分CHD的一个因素 环境或基因。虽然我们的团队和其他人最近开发了非常有用的光学 成像工具（例如，光学相干断层扫描- OCT），以评估血流动力学和剪切应力，以及 将这些测量与CHD联系起来，很难将剪切应力与受影响的分子联系起来， 途径。我们的小组和其他人已经对控制和剪切应力扰动进行了qPCR实验 研究异常的血液动力学如何改变基因表达。然而，这种方法需要整个 胚胎心脏的一次测量，失去了所有的空间和细胞类型的信息，特别是在 内胚层为了成功地评估剪切应力如何影响整个细胞的分子信号传导， 循环心脏，我们需要改进我们的OCT方法，开发评估胚胎心脏的3D方法 基因表达，并创建一个先进的图像处理管道来分析数据和相关的区域剪切 胁迫对基因表达的影响。 这项更新建议将继续我们的工作，开发工具，可以导致一个更复杂的 了解心脏功能如何（例如，血流动力学和电脉冲传导）影响心脏 开发，使潜在的治疗，以避免或减轻冠心病。在本提案中，我们将重点开发 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CHD。在我们的初步研究中，我们增加了血液流动（导致 增加的OSI），以表明OSI的改变导致较小的心脏垫（瓣膜前体）， 最后是冠心病。在我们的两种疾病模型中，存在增加的顺应性血流量和较小的缓冲垫 （胎儿酒精谱系障碍- FASD;腭心面综合征/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，并大大降低发病率和CHD。 我们的具体目标包括：1）推进我们的OCT系统和剪切应力分析，2）开发荧光， 原位杂交（FISH）协议以测量3D中的基因表达，3）开发图像处理流水线 将基因表达与剪切应力相关联，以及4）确定剪切应力对基因表达的影响。后 完成后，我们将有更多关于剪切应力如何影响分子表达的信息。与 有了这些知识，我们将更好地确定哪些分子途径受改变的影响最大。 血流动力学，开发早期检测方法，并可能开发预防CHD的策略， 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.