Telomerase Reverse Transcriptase in Vascular Homeostasis

血管内皮在协调多种循环系统中起着至关重要的作用 血液流动、血栓形成、白细胞运输甚至新陈代谢等功能。正常 内皮功能的特征在于非增殖的静止细胞表型， 非迁移性，并表现出细胞表面，防止血栓形成，炎症和脂质 沉积，从而抗动脉粥样硬化和血管疾病。一把稳定的钥匙 内皮静止的刺激是细胞上的层流剪切应力（FSS 表面是直血管段的特征。相比之下，弯曲和 分支动脉经历混乱的FSS，称为扰动流，其指示较少的 稳定的、活化的、内皮细胞表型，更易发生动脉粥样硬化。的 控制内皮细胞表型对流体剪切应力的反应的机制是 不完全理解。在本申请中，我们提供的数据表明，过氧化物酶体增殖物 γ辅激活因子-1 α（PGC 1 α）是内皮细胞中的流体剪切应力反应因子， 上调与层流，但不是振荡FSS。PGC 1 α的上调对于 激活与正常血管稳态相关的关键途径，如Klf 2、Notch和 促进稳定抗动脉粥样硬化内皮表型的eNOS。激动人心的飞行员数据 将这种效应与端粒酶逆转录酶（TERT）的上调及其 核外，端粒长度不依赖，功能稳定维持 线粒体稳态响应层状FSS。缺乏TERT活性的内皮细胞 显示线粒体碎片，并未能与流动对齐，这是一个关键的功能， 来抵抗动脉粥样硬化。总的来说，这些数据提示了我们的中心假设， 内皮PGC 1 α-TERT信号是内皮和血管适应 剪切和正常血管稳态。为了研究这一假设，我们建议首先 用一种新的方法确定PGC 1 α如何影响体内内皮对FSS的反应， 他莫昔芬诱导的Cre/Lox系统产生内皮特异性PGC 1 α基因切除， 原位共聚焦显微镜、单细胞RNA-seq、Network Medicine和ApoE-/-动脉粥样硬化 模型类似地，我们将使用与诱导型内皮细胞TERT基因切除相同的策略。 最后，使用缺乏PGC 1 α或TERT的细胞培养物，我们将解剖 PGC 1 α-TERT信号传导影响内皮细胞FSS反应性的机制， 特别关注FSS诱导的PGC 1 α基因组占用，线粒体和细胞代谢， 内皮细胞流动排列，和TERT定位于细胞核相对于线粒体。 总的来说，这些研究将提供一个新的范式，内皮细胞 对FSS的反应性和维持静止的内皮单层的要求， 抵抗血管疾病。有了这些信息，我们应该有必要的洞察力来设计新的 减少血管疾病的发病率和死亡率。

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.