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：
10412985
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 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10412985
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 Cre lox recombination system 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 Network-based Nuclear Pathway interactions Peroxisome Proliferators Phenotype Physiological Play Prevention Prevention therapy Proteins RNA-Directed DNA Polymerase Role Signal Transduction Solid Stimulus 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 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.

血管内皮在协调潜水电路中起着至关重要的作用血流，血栓形成，白细胞运输甚至代谢等功能。普通的内皮功能的特征是静态细胞表型，该表型未增殖，非移民，并表现出可防止血栓形成，注射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。钥匙稳定内皮静止的刺激是细胞上的层流流体剪应力（FSS）表面是直血段的特征。相反，弯曲和分支动脉经历混乱的FSS，称为干扰流动，这决定了较少的稳定，激活，内皮表型，更容易受到动脉粥样硬化的影响。这响应液体剪切应力的内皮表型的机制是不完全理解。在此应用程序中，我们介绍了过氧化物组增殖物的数据伽马共激活剂1α（PGC1α）是内皮中的流体剪切应力响应因子，用层流上调，但没有振荡的FSS。 PGC1α的上调对于与正常血管稳态有关的关键途径的激活，例如KLF2，Notch和促进稳定的抗动物性内皮表型的eNOS。令人兴奋的飞行员数据将此效果与端粒酶逆转录酶（TERT）及其的上调联系起来核外，端粒长度无关，稳定维护的功能线粒体稳态应对层流FSS。缺乏TERT活性的内皮显示线粒体碎片，无法与Flow保持一致，这是需要的关键功能这些数据共同促使我们的中心假设是内皮和血管适应至需要的内皮PGC1α-TERT信号传导剪切和正常的血管稳态。为了研究这一假设，我们提议首先确定PGC1α如何使用A影响体内FSS的内皮反应他莫昔芬可诱导的Cre/Lox系统产生内皮特异性PGC1α基因切除，在原位共聚焦显微镜，单细胞RNA-seq，网络药物和APOE - / - 动脉粥样硬化模型。同样，我们将使用相同的策略和可诱导的内皮TERT基因切除。最后，使用缺乏PGC1α或TERT的细胞的细胞培养，我们将剖析 PGC1α-TERT信号传导影响内皮FSS反应性的机制特别关注FSS诱导的PGC1α基因组占用率，线粒体和细胞代谢，内皮细胞的流动比对，并将其定位于细胞核与线粒体。总的来说，这些研究将洞悉内皮细胞的新范式对FSS的响应以及维持静止的内皮单层的要求抵抗血管疾病。有了这些信息，我们应该有必要的见解来设计新的减轻血管疾病发病率和死亡率的疗法。