Shear stress, endothelial miRNAs, and AV calcification

描述（由申请方提供）：钙化性主动脉瓣（AV）疾病是全球心脏相关死亡的主要原因，也是其他心血管事件的强风险因素。AV疾病最初被认为是一个“磨损”过程，但现在已知它是一种导致硬化和钙化病变的活动性炎症性疾病。此外，AV疾病优先发生在纤维侧，而心室侧保持相对健康。剪切导致这种疾病的机制仍然未知。我们假设，干扰的流动条件存在于纤维瘤侧，并调节内皮基因表达-导致AV炎症和钙化的miRNA和mRNA。在此之前，我们已经在人主动脉瓣内皮细胞（HAVEC）中鉴定了剪切反应性miRNA;然而，这些miRNA的重要性和功能仍然未知。最近，我们已经从猪AV中新鲜分离的内皮富集RNA中鉴定了侧依赖性miRNA。从这项工作中，我们已经确定miR-199a-5p，miR-214和miR-486 - 5p为前三名剪切和/或侧依赖性miRNA。我们假设这些是内皮细胞中的机械敏感性miRNA，导致AV炎症和钙化。为了解决这一假设，我们提出了以下目标。首先，我们将确定机械敏感性miRNA在体外和猪AV中的主动脉瓣内皮中的作用。将使用qPCR和原位杂交在培养的AVEC和新鲜分离的来自人和猪AV的内皮富集RNA中验证miR-199a-5p、214和486 - 5p的剪切和侧向依赖性表达。这些miRNA在剪切敏感性内皮功能中的作用，包括炎症、迁移、凋亡、增殖和细胞周期，将使用功能获得或丧失研究来研究。接下来，我们将使用猪AV体外确定机械敏感性miRNA在主动脉瓣内皮中的作用。将在暴露于层流剪切或振荡剪切应力条件下的猪AV中完成具有验证的阵列研究，以鉴定内皮富集RNA以及来自剩余组织（主要是间质细胞）的RNA中的剪切和侧依赖性miRNA。验证后，将调节关键miRNA（miR-199a、214和486 - 5p）的水平，并评估与炎症和钙化有关的细胞功能变化。最后，我们将确定机械敏感性miRNA在体内AV钙化和炎症中的作用。AV疾病将使用喂食西方饮食的ApoE-/-小鼠在体内建模。将表征炎症、硬化（瓣膜厚度）、细胞凋亡和钙化的发展。该AV钙化的小鼠模型将用于使用LNA抑制剂或miRNA前体在体内环境中验证来自目的1和2的所选miRNA。这些miRNAs的鉴定将为AV疾病发生的分子机制提供深入了解，提供潜在的治疗靶点和诊断标志物，以及设计组织工程瓣膜的重要考虑因素。

DESCRIPTION (provided by applicant): Calcific aortic valve (AV) disease is a major cause of cardiac-related deaths worldwide and is a strong risk factor for additional cardiovascular events. AV disease was originally thought to be a "wear and tear" process but now it is known to be an active inflammatory disease leading to sclerotic and calcific lesions. Further, AV disease occurs preferentially on the fibrosa side whereas the ventricularis side remains relatively healthy. The mechanism by which shear contributes to this disease remains unknown. We hypothesize that disturbed flow conditions are present on the fibrosa side and regulate endothelial gene expression - miRNAs and mRNAs that lead to AV inflammation and calcification. Previously, we have identified shear-responsive miRNAs in human aortic valvular endothelial cells (HAVECs); however, the importance and function of these miRNAs remains unknown. Recently, we have identified side-dependent miRNAs from freshly isolated endothelial- enriched RNA in porcine AVs. From this work, we have identified miR-199a-5p, miR-214, and miR-486-5p as the top three shear- and/or side-dependent miRNAs. We hypothesize that these are mechanosensitive miRNAs in endothelium which leads to AV inflammation and calcification. To address this hypothesis, we propose the following aims. First, we will determine the role of mechanosensitive miRNAs in aortic valve endothelium in vitro and in porcine AVs. Shear- and side-dependent expression of miR-199a-5p, 214, and 486- 5p will be validated in cultured AVECs and freshly isolated, endothelial-enriched RNA from human and porcine AVs using qPCR and in situ hybridization. The role of these miRNAs in shear-sensitive endothelial function, including inflammation, migration, apoptosis, proliferation, and cell cycle will be investigated using gain- r loss-of-function studies. Next, we will determine the role of mechanosensitive miRNAs in aortic valve endothelium ex vivo using porcine AVs. Array studies with validation will be completed in porcine AVs exposed to laminar shear or oscillatory shear stress conditions to identify shear- and side-dependent miRNAs in both endothelial-enriched RNA as well as RNA from the leftover tissue (mainly interstitial cells). Following validation, the levels of the key miRNAs (miR-199a, 214, and 486-5p) will be modulated and assessed for cell function changes pertaining to inflammation and calcification. Finally, we will determine the role of mechanosensitive miRNAs in AV calcification and inflammation in vivo. AV disease will be modeled in vivo using ApoE-/- mice fed a Western diet. Development of inflammation, sclerosis (valve thickness), apoptosis, and calcification will be characterized. This mouse model of AV calcification will be used to validate the selected miRNAs from Aims 1 and 2 in an in vivo setting with the use of LNA inhibitors or miRNA precursors. Identification of these miRNAs will provide insight to molecular mechanisms by which AV disease occurs, offering potential therapeutic targets and diagnostic markers as well as important considerations in designing tissue-engineered valves.