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 i nclude 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.
Abnormal blood flow has been shown to play a role in the formation of congenital heart defects (CHDs). However, few tools exist to study how forces exerted by blood flow early in heart development result in altered molecular signaling. Our proposal centers on creating optical imaging to investigate mechanical stresses experienced by cells lining the beating, early-stage heart tube and how these cells and adjacent tissues respond in gene expression in each area of the heart during normal, diseased, and rescued conditions.
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