Shear stress, endothelial miRNAs, and AV calcification

剪切应力、内皮 miRNA 和 AV 钙化

基本信息

  • 批准号:
    8720061
  • 负责人:
  • 金额:
    $ 45.99万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
说明(申请人提供):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烈危险因素。房室疾病最初被认为是一种“磨损”的过程,但现在人们知道它是一种活动性炎症性疾病,导致硬化性和钙化病变。此外,房室疾病优先发生在纤维侧,而室侧则相对健康。剪切力导致这种疾病的机制尚不清楚。我们假设纤维侧存在血流紊乱,并调节内皮基因表达--导致房室炎症和钙化的miRNAs和mRNAs。以前,我们已经在人主动脉瓣内皮细胞(HAVECs)中发现了剪切反应miRNAs;然而,这些miRNAs的重要性和功能仍不清楚。最近,我们从新鲜分离的猪动静脉内皮细胞丰富的RNA中鉴定出了侧向依赖的miRNAs。从这项工作中,我们确定miR-199a-5p、miR-214和miR-486-5p是前三个剪切和/或侧向依赖的miRNAs。我们推测这些是内皮细胞中机械敏感的miRNAs,导致房室炎症和钙化。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miRNAs在体外主动脉瓣内皮细胞和猪动静脉内皮细胞中的作用。利用qPCR和原位杂交技术,将在培养的动静脉内皮细胞以及新鲜分离的人和猪动静脉动静脉内皮细胞中验证miR-199a-5p、214和486-5p的剪切和侧向依赖表达。这些miRNAs在剪切力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,将通过Gain-r功能丧失研究来研究。接下来,我们将使用猪动静脉系统来确定机械敏感的miRNAs在体外主动脉瓣内皮细胞中的作用。经过验证的阵列研究将在暴露于层流剪切或振荡剪切应力条件下的猪动静脉动静脉中完成,以识别内皮丰富的RNA以及残留组织(主要是间质细胞)中的剪切和侧向依赖的miRNAs。在验证之后,关键的miRNAs(miR-199a、214和486-5p)的水平将被调节和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miRNAs在体内房室钙化和炎症中的作用。将使用喂食西方饮食的ApoE-/-小鼠在体内建立AV疾病的模型。炎症、硬化(瓣膜厚度)、细胞凋亡和钙化的发展将是其特征。这种房室钙化的小鼠模型将用于在使用LNA抑制剂或miRNA前体的体内环境中验证从AIMS 1和2中选择的miRNAs。这些miRNAs的识别将有助于深入了解AV疾病发生的分子机制,提供潜在的治疗靶点和诊断标记,以及设计组织工程瓣膜时的重要考虑因素。

项目成果

期刊论文数量(0)
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Hanjoong Jo其他文献

Hanjoong Jo的其他文献

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{{ truncateString('Hanjoong Jo', 18)}}的其他基金

Role of CEBPb in flow-dependent endothelial dysfunction and atherosclerosis
CEBPb 在血流依赖性内皮功能障碍和动脉粥样硬化中的作用
  • 批准号:
    10638650
  • 财政年份:
    2023
  • 资助金额:
    $ 45.99万
  • 项目类别:
HEG1 in endothelial function and atherosclerosis
HEG1在内皮功能和动脉粥样硬化中的作用
  • 批准号:
    10272942
  • 财政年份:
    2021
  • 资助金额:
    $ 45.99万
  • 项目类别:
HEG1 in endothelial function and atherosclerosis
HEG1在内皮功能和动脉粥样硬化中的作用
  • 批准号:
    10630328
  • 财政年份:
    2021
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    10171094
  • 财政年份:
    2020
  • 资助金额:
    $ 45.99万
  • 项目类别:
Role of flow-sensitive KLK10 in endothelial dysfunction and atherosclerosis
流量敏感的 KLK10 在内皮功能障碍和动脉粥样硬化中的作用
  • 批准号:
    10210428
  • 财政年份:
    2018
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    8563026
  • 财政年份:
    2013
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    10510621
  • 财政年份:
    2013
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    9063173
  • 财政年份:
    2013
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    10321908
  • 财政年份:
    2013
  • 资助金额:
    $ 45.99万
  • 项目类别:
Shear stress, endothelial miRNAs, and AV calcification
剪切应力、内皮 miRNA 和 AV 钙化
  • 批准号:
    9270596
  • 财政年份:
    2013
  • 资助金额:
    $ 45.99万
  • 项目类别:

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