Telomerase Reverse Transcriptase in Vascular Homeostasis

端粒酶逆转录酶在血管稳态中的作用

基本信息

批准号：
10619665
负责人：
John Francis Keaney
金额：
$ 59.47万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-06 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10619665
关键词：
Aging Animals Aorta Aortic Segment Apolipoprotein E Arteries Atherosclerosis Binding Blood Vessels Blood flow Cell Culture Techniques Cell Nucleus Cell surface Cells Confocal Microscopy Cultured Cells Data Dependence Deposition Descending aorta Elements Endothelial Cells Endothelium Excision Exhibits Exposure to Genes Genetic Genetic Transcription Genome Glucose Homeostasis Human Immunofluorescence Immunologic In Situ Inflammation Knock-out Knowledge Length Leukocyte Trafficking Link Lipids Liquid substance Maps Medicine Metabolism Mitochondria Modeling Morbidity - disease rate Morphology Mus Nuclear Pathway interactions Peroxisome Proliferators Phenotype Physiological Play Predisposition Prevention Prevention therapy Proteins RNA-Directed DNA Polymerase Role Signal Transduction Solid Stimulus System Tamoxifen Telomerase Testing Thrombosis Transgenic Mice Up-Regulation Vascular Diseases Vascular Endothelium Work aortic arch biological adaptation to stress design experience experimental study fatty acid oxidation improved in vivo inducible Cre insight loss of function monolayer mortality notch protein novel therapeutics overexpression preservation prevent response shear stress single-cell RNA sequencing telomere therapy design transcriptome sequencing

项目摘要

The vascular endothelium plays an essential role in coordinating diverse circulatory functions such as blood flow, thrombosis, leukocyte trafficking, and even metabolism. Normal endothelial function is characterized by a quiescent cell phenotype that is non-proliferative, non-migratory, and exhibits a cell surface that prevents thrombosis, inflammation, and lipid deposition, thereby resisting atherosclerosis and vascular disease. A key stabilizing stimulus for endothelial quiescence is laminar fluid shear stress (FSS) on the cell surface that is a feature of straight vascular segments. In contrast, curved and branching arteries experience chaotic FSS, called disturbed flow, that dictates a less stable, activated, endothelial phenotype that is more susceptible to atherosclerosis. The mechanisms governing endothelial phenotype in response to fluid shear stress are incompletely understood. In this application, we present data that peroxisome proliferator gamma coactivator-1α (PGC1α), is a fluid shear stress-responsive factor in endothelium that is upregulated with laminar, but not oscillatory FSS. Upregulation of PGC1α is important for the activation of key pathways linked to normal vascular homeostasis such as Klf2, Notch, and eNOS that promote a stable anti-atherosclerotic endothelial phenotype. Exciting pilot data links this effect to upregulation of telomerase reverse transcriptase (TERT) and its extra-nuclear, telomere length-independent, function to stabilize maintain mitochondrial homeostasis in response to laminar FSS. Endothelium lacking TERT activity shows mitochondrial fragmentation and fails to align with flow, a key function needed to resist atherosclerosis. Collectively, these data prompt our central hypothesis that endothelial PGC1α-TERT signaling is required for endothelial and vascular adaptation to shear and normal vascular homeostasis. To investigate this hypothesis, we propose to first determine how PGC1α influences endothelial responses to FSS in vivo using a tamoxifen-inducible Cre/Lox system producing endothelial specific PGC1α-gene excision, in situ confocal microscopy, single-cell RNA-seq, Network Medicine, and the ApoE-/- atherosclerosis model. Similarly, we will use the same strategy with inducible endothelial TERT gene excision. Finally, using cell culture of cells lacking either PGC1α or TERT, we will dissect the mechanisms whereby PGC1α-TERT signaling impacts endothelial FSS responsiveness with a particular focus on FSS-induced PGC1α genome occupancy, mitochondrial and cellular metabolism, endothelial cell flow alignment, and TERT localization to the nucleus vs. mitochondria. Collectively, these studies will provide insight into a new paradigm of endothelial cell responsiveness to FSS and the requirements to maintain a quiescent endothelial monolayer that resists vascular disease. With this information, we should have the requisite insight to design new therapies to alleviate morbidity and mortality from vascular disease.

血管内皮在协调多种循环系统中发挥着重要作用血流、血栓形成、白细胞运输、甚至新陈代谢等功能正常。内皮功能的特征是非增殖的静止细胞表型，非迁移性，并具有防止血栓形成、炎症和脂质的细胞表面沉积，从而抵抗动脉粥样硬化和血管疾病的关键。内皮静止的刺激是细胞上的层流流体剪切应力 (FSS) 表面是直维管段的特征。分支动脉经历混乱的 FSS，称为扰动血流，这决定了较少的稳定、活化的内皮表型更容易发生动脉粥样硬化。控制内皮表型响应流体剪切应力的机制是在此应用中，我们提供了过氧化物酶体增殖剂的数据。 γ 辅激活因子-1α (PGC1α) 是内皮细胞中的一种流体剪切应力响应因子， PGC1α 的上调对于层流 FSS 很重要，但不是振荡 FSS。激活与正常血管稳态相关的关键通路，例如 Klf2、Notch 和 eNOS 促进稳定的抗动脉粥样硬化内皮表型令人兴奋的试验数据。将这种效应与端粒酶逆转录酶 (TERT) 的上调及其相关性联系起来核外、端粒长度无关、稳定维持功能线粒体稳态响应缺乏 TERT 活性的层流内皮细胞。显示线粒体碎片并且无法与流量对齐，这是所需的关键功能总的来说，这些数据提示了我们的中心假设：内皮和血管适应需要内皮 PGC1α-TERT 信号传导为了研究这一假设，我们建议首先确定 PGC1α 如何影响体内内皮细胞对 FSS 的反应他莫昔芬诱导的 Cre/Lox 系统产生内皮特异性 PGC1α 基因切除，原位共聚焦显微镜、单细胞 RNA-seq、网络医学和 ApoE-/- 动脉粥样硬化同样，我们将使用与诱导型内皮 TERT 基因切除相同的策略。最后，使用缺乏 PGC1α 或 TERT 的细胞培养物，我们将剖析 PGC1α-TERT 信号传导影响内皮 FSS 反应的机制特别关注 FSS 诱导的 PGC1α 基因组占用、线粒体和细胞代谢，内皮细胞流排列，以及 TERT 定位到细胞核与线粒体。总的来说，这些研究将为内皮细胞的新范式提供见解对 FSS 的反应性以及维持静止内皮单层的要求有了这些信息，我们就应该拥有设计新产品所需的洞察力。减轻血管疾病发病率和死亡率的治疗。