Biomechanical regulation of valvulogenesis

描述(申请人提供)：瓣膜相关先天性心脏病(CHD)是导致早产和婴儿死亡的主要原因。尽管研究发现了心脏形态发生的重要遗传调节因素，但由于缺乏对这些途径如何协调驱动瓣膜形成和重塑的了解，这些途径转化为临床益处一直受到阻碍。随着胚胎瓣膜的生长和成熟，它们会暴露在越来越苛刻的机械环境中，但这些力量的功能后果还远未被了解。瓣膜的发生也可以被认为是一个工程过程，而不是一个纯粹的遗传程序。因此，揭示这些新的支配关系是至关重要的，但由于缺乏分析工具和实验方法来分离和量化活的胚胎瓣膜的机械效应，因此一直具有挑战性。首先，我们开发了独特的生物力学测试设备，可以量化胚胎坐垫和瓣膜的生物力学。其次，我们创建了一种新的斑点跟踪算法，与高频超声相结合，可以非侵入性地量化胚胎瓣膜内的动态组织应变。第三，我们开发了一种独特的有限元模拟策略，该策略与活体测量迭代，以在解剖上精确的几何图形中映射局部生物力学参数。第四，我们创造了第一个实验策略，通过飞秒激光消融(FLP)非侵入性地在活体禽类胚胎中制造局部隔离的心脏内缺陷。利用这些使能技术，我们这项提案的目标是识别和描述指导胚胎瓣膜形成的机械遗传学关系。我们的总体假设是，机械信号通过同时调节多个瓣膜形成信号通路来协调胚胎瓣膜的塑形和加强。我们将首先量化瓣膜周围不断变化的体内机械环境及其生物力学适应(目标1)。接下来，我们将确定已知的瓣膜形成分子调控网络如何在体外受到机械刺激的调节(目标2)。然后，我们在活体内使用局部控制的光消融禽类胚胎瓣膜来测试先前实验所提出的机械遗传学机制(目标3)。这一提议将产生关于胚胎瓣膜形成过程中体内生物力学环境的重要量化细节，以及这如何调节局部基因表达以促进瓣膜形成和成熟。这些新的信息将补充现有的基因操作研究数据集，并指导对这些结果的新解释。更好地了解指导瓣膜形成和重塑的力学生物学关系将极大地拓宽解释CHD发病机制的机制，并使新的临床策略能够预防或修复CHD。

DESCRIPTION (provided by applicant): Valve related congenital heart defects (CHD) are a major cause of preterm and infant death. Despite considerable research progress uncovering important genetic regulators of cardiac morphogenesis, translation to clinical benefit has been hampered by a lack of understanding of how these pathways coordinate to drive valve formation and remodeling. The embryonic valves are exposed to an increasingly demanding mechanical environment as they grow and mature, but the functional consequences of these forces are far less understood. Valvulogenesis can alternatively be considered an engineering process rather than a purely genetic program. Uncovering these new governing relationships is therefore essential but has been challenging because of the lack of analytical tools and experimental approaches that can isolate and quantify mechanical effects in live embryonic valves. First, we have developed unique biomechanical testing devices that can quantify embryonic cushion and valve biomechanics. Second, we have created a novel speckle tracking algorithm in conjunction with high frequency ultrasound that can non-invasively quantify dynamic tissue strains within the embryonic valves. Third, we developed a unique finite element simulation strategy that iterates with in vivo measurements to map local biomechanical parameters in anatomically precise geometries. Fourth, we have created the first experimental strategy to create locally isolated intracardiac defects in live avian embryos non-invasively through femtosecond laser photoablation (FLP). Using these enabling technologies, our objective for this proposal is to identify and characterize mechano-genetic relationships that guide the formation of the embryonic valves. Our overall hypothesis is that mechanical signaling orchestrates the sculpting and strengthening of the embryonic valves through simultaneous regulation of multiple valvulogenic signaling pathways. We will first quantify the changing in vivo mechanical environments surrounding the valves and their biomechanical adaptation (Aim 1). We will next determine how the network of known molecular regulators of valvulogenesis is modulated by mechanical stimulation in vitro (Aim 2). Then we test the mechano-genetic mechanisms suggested by the previous experiments in vivo using locally controlled photoablations to avian embryonic valves (Aim 3). This proposal will generate significant quantitative detail of the in vivo biomechanical environment during embryonic valvulogenesis and how this regulates local gene expression to promote valve formation and maturation. This novel information will complement existing datasets from genetic manipulation studies and guide new interpretations of these results. A better understanding of the mechanobiological relationships guiding valve formation and remodeling significantly broadens the array of mechanisms to explain the pathogenesis of CHD and tools to enable new clinical strategies to prevent or repair CHD.