Shear stress, endothelial miRNAs, and AV calcification

剪切应力、内皮 miRNA 和 AV 钙化

• 批准号: 8563026
• 负责人: Hanjoong Jo
• 金额: $ 46.21万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-12 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8563026
• 关键词: Address; Apolipoprotein E; Apoptosis; Cardiac; Cardiovascular system; Cell Cycle; Cell physiology; Cessation of life; Cryoultramicrotomy; Development; Diagnostic; Diet; Disease; Endothelial Cells; Endothelium; Event; Family suidae; Gene Expression; Human; In Situ Hybridization; In Situ Nick-End Labeling; In Vitro; Inflammation; Inflammatory; Lead; Lesion; Messenger RNA; MicroRNAs; Molecular; Mus; Process; RNA; Risk Factors; Role; Sclerosis; Side; Staining method; Stains; Stress; Thick; Tissue Engineering; Tissues; Validation; Western Blotting; Work; aortic valve; aortic valve disorder; calcification; design; feeding; in vivo; in vivo Model; inhibitor/antagonist; insight; interstitial cell; loss of function; migration; mouse model; pre-miRNA; public health relevance; shear stress; therapeutic target

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.

描述（由申请人提供）：钙化主动脉瓣（AV）疾病是全球与心脏相关死亡的主要原因，并且是额外心血管事件的强大风险因素。 AV疾病最初被认为是“磨损”过程，但现在众所周知，它是一种活跃的炎症性疾病，导致硬化性和钙化病变。此外，AV疾病优先发生在纤维侧，而心室侧仍然相对健康。剪切导致这种疾病的机制仍然未知。我们假设纤维侧存在流动条件，并调节内皮基因表达 - 导致AV炎症和钙化的miRNA和mRNA。以前，我们已经确定了人主动脉瓣膜内皮细胞（HAVEC）中的剪切响应性miRNA。但是，这些miRNA的重要性和功能仍然未知。最近，我们从猪AV中鉴定出刚分离的内皮富含内皮的RNA的侧依赖性miRNA。从这项工作中，我们将miR-199a-5p，miR-214和miR-486-5p确定为前三名剪切和/或侧依赖的miRNA。我们假设这些是内皮中的机械敏感miRNA，导致AV炎症和钙化。为了解决这一假设，我们提出以下目的。首先，我们将确定机械敏感的miRNA在体外和猪AV中的主动脉瓣内皮中的作用。 MiR-199a-5p，214和486-5p的剪切和侧面依赖性表达将在培养的AVEC中得到验证，并使用QPCR和原位杂交在培养的AVEC和新鲜分离的，内皮的富含内皮的RNA中得到验证。这些miRNA在剪切敏感的内皮功能中的作用，包括炎症，迁移，凋亡，增殖和细胞周期，将使用功能丧失研究研究。接下来，我们将使用猪AVS确定机械敏感的miRNA在主动脉瓣内皮中的作用。具有验证的阵列研究将在暴露于层状剪切或振荡性剪切应力条件下的猪AV中完成，以鉴定内皮增添富含内皮的RNA的剪切和侧依赖性miRNA，以及剩余组织（主要是间隙细胞）的RNA。验证后，将调制关键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的miRNA在体内的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。炎症，硬化症（瓣膜厚度），凋亡和钙化的发展将被表征。这种AV钙化的小鼠模型将使用使用LNA抑制剂或miRNA前体在体内环境中从AIMS 1和2中验证所选的miRNA。这些miRNA的鉴定将为您的AV疾病发生的分子机制提供见识，从而提供潜在的治疗靶标和诊断标记以及设计组织工程瓣膜的重要考虑因素。